AU2022204080B2 - Induction of pluripotent cells - Google Patents

Abstract

#$%^&*AU2022204080B220250717.pdf##### INDUCTION OF PLURIPOTENT CELLS ABSTRACT The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical 5 applications. Here we describe a chemical approach that dramatically improves (>200 fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells. INDUCTION OF PLURIPOTENT CELLS 10 Jun 2022 ABSTRACT The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical 5 applications. Here we describe a chemical approach that dramatically improves (>200 fold) the 2022204080 efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells.

Description

INDUCTION OF INDUCTION OF PLURIPOTENT CELLS PLURIPOTENT CELLS CROSS-REFERENCESTOTORELATED CROSS-REFERENCES RELATED APPLICATIONS APPLICATIONS

[0001] This

[0001] This application application thethe claims claims benefitunder benefit U.S.C.§ 1.119(e) under3535U.S.C. of of § 1.119(e) U.S. provisional U.S.provisional

2022204080 Application No. 61/252,548, Application No. 61/252,548,filed filed October October16, 16,2009, 2009,the thecontents contents of ofwhich whichare areincorporated incorporated by reference by referenceininthetheentirety. entirety.This This application application is a is a divisional divisional of AU of AU 2019216658, 2019216658, which is a which is a divisional of divisional of AU 2017201542, AU 2017201542, which which is is a divisionalofofAUAU a divisional 2014250681, 2014250681, which which is a divisional is a divisional

of AU 2010306627, AU 2010306627, which which is the is the national national phase phase entry entry of of PCT/US2010/052896, PCT/US2010/052896, the entire the entire

contents contents ofofeach eachof of which which are incorporated are incorporated by reference by reference in the entirety. in the entirety.

BACKGROUND BACKGROUND OFOFTHE THE INVENTION INVENTION

[0002] Recent

[0002] Recent advances advances in generating in generating human human induced induced pluripotent pluripotent stem cells stem cells (iPSCs) (iPSCs)

(Takahashi, (Takahashi, K. K. et et al.,Cell al., Cell 131, 131, 861-72 861-72 (2007); (2007); Yu, J.Yu, J. etScience et al., al., Science 318, 1917-20 318, 1917-20 (2007); (2007); Muller,L.U.W., Muller, L.U.W., et al., et al., Mol. Mol. Ther. Ther. 17, 947-53 17, 947-53 (2009)) (2009)) have have raised raised hopes for hopes for their their utility in utility in biomedical research biomedical research and andclinical clinical applications. applications. However, iPSC However, iPSC generation generation is is still aa very still very slow slow

(~4 weeks) (~4 weeks) andand inefficient inefficient (<0.01% (<0.01% (Takahashi, (Takahashi, K. Cell K. et al., et al., 131,Cell 131,(2007); 861-72 861-72Yu, (2007); J. et Yu, J. et al., al., Science 318,1917-20 Science 318, 1917-20 (2007)) (2007)) process process that results that results in a heterogeneous in a heterogeneous populationpopulation of cells. of cells.

Identifying fully Identifying fully reprogrammed iPSCs reprogrammed iPSCs from from such such a mixture a mixture is tedious,and is tedious, and requiresspecific requires specific expertise ininhuman expertise human pluripotent pluripotent cell cell culture. culture.

[0003] the the Although

[0003] Although dangers dangers of genomic of genomic insertion insertion of exogenous of exogenous reprogramming factorsfactors reprogramming is is being overcome, being overcome,the thelow lowefficiency efficiencyand andslow slowkinetics kineticsof ofreprogramming reprogramming continue continue to present to present a a formidable problemfor formidable problem forultimate ultimate applications applications of of human humaniPSC. iPSC. ForFor example, example, an increase an increase in in

genetic or epigenetic epigenetic abnormalities abnormalities could occur during during the the reprogramming process,where reprogramming process, where tumor suppressors tumor suppressorsmay maybebeinhibited inhibitedand andoncogenic oncogenic pathways pathways may may be activated. be activated. Though Though

recent studies studies have have reported reported an an improved efficiency of improved efficiency of reprogramming reprogramming by by genetic genetic

manipulations manipulations (Feng, (Feng, B.al., B. et et al., CellCell StemStem Cell Cell 4, 4, 301-12 301-12 (2009)) (2009)) in addition in addition to the to the original original four factors, factors,such such manipulations manipulations typically typically make the process even make the morecomplex even more complexandand increase increase

the risk the risk of of genetic geneticalterations alterationsandand tumorigenicity. tumorigenicity. Thus, Thus, there there is stillisa still a tremendous tremendous need need for a for a safer, easier and safer, easier andmore more efficient efficient procedure procedure for human for human iPSC generation iPSC generation and facilitate and facilitate

identifying identifying and and characterizing characterizing fundamental mechanisms fundamental mechanisms of of reprogramming. reprogramming.

1

[0003a] Any

[0003a] Any discussion discussion of of theprior the priorart art throughout throughoutthe thespecification specification should should in in no waybebe no way 25 Jun 2025 2022204080 25 Jun 2025

considered as an considered as an admission admissionthat that such such prior prior art art is iswidely widelyknown or forms known or forms part part of of the the common common

general knowledge general knowledge in field. in the the field.

[0003b] Unlessthe

[0003b] Unless thecontext contextclearly clearlyrequires requires otherwise, otherwise, throughout throughoutthe thedescription description and andthe the 55 claims, claims, thethe words words “comprise”, "comprise", “comprising”, "comprising", andlike and the the like are are to be to be construed construed in an in an inclusive inclusive

sense asopposed sense as opposed to exclusive to an an exclusive or exhaustive or exhaustive sense; sense; that is that is to to say, in say, in theofsense of the sense

“including, butnotnot "including, but limited limited to”. to". 2022204080

BRIEF SUMMARY BRIEF SUMMARY OFOFTHE THEINVENTION INVENTION

[0004]

[0004] InIna afirst firstaspect, aspect,the thepresent present invention invention provides provides an in an in or vitro vitro or exmethod ex vivo vivo method of of 10 10 inducing inducing non-pluripotent non-pluripotent mammalian mammalian cells cells into induced into induced pluripotent pluripotent stem stem cells, cells, comprising: comprising:

introducing intothethe introducing into non-pluripotent non-pluripotent mammalian mammalian cells at cells least at twoleast two transcription transcription factors factors selected from selected from the the group group consisting consisting of:Oct-3/4 of: (i) (i) Oct-3/4 andandKlf; and Klf; (ii)and (ii) Oct-3/4 Oct-3/4 and and one or one more of or more of

Klf, Sox2, Klf, Sox2, and c-Myc;and and c-Myc; and contacting the non-pluripotent contacting the non-pluripotent mammalian cellswith mammalian cells withananALK5 ALK5 inhibitor, inhibitor, a MEK a MEK

15 15 inhibitor,andand inhibitor, a ROCK a ROCK inhibitor, inhibitor, wherein wherein the the ROCKROCK inhibitor inhibitor is present is present in aninamount an amount effective effective to toincrease increaseefficiency efficiencyofof reprogramming reprogramming aa non-pluripotent mammalian non-pluripotent mammalian celltotoa a cell

pluripotent cell, pluripotent cell,

whereinthe wherein the non-pluripotent non-pluripotentmammalian mammalian cells cells areare culturedininthe cultured theabsence absenceofoffeeder feeder cells cells and underconditions and under conditions sufficient sufficient to induce to induce pluripotent pluripotent stem cells. stem cells.

20 [0004a] 20 [0004a] In a second In a second aspect, aspect, the present the present invention invention provides provides a mixture a mixture comprising: comprising:

(a) (a) oneone or or more more mammalian mammalian cells; cells;

(b) (b) a Rho a Rho kinase kinase (ROCK) (ROCK) inhibitor inhibitor

(c) (c) an an ALK5 ALK5 inhibitor;and inhibitor; and (d) (d) a MAP/ERK a MAP/ERK kinase kinase (MEK) (MEK) inhibitor; inhibitor;

25 25 whereinthe wherein the ROCK ROCK inhibitor inhibitor is is presentininananamount present amount effectivetotoincrease effective increaseefficiency efficiency of of reprogramming a non-pluripotent reprogramming a non-pluripotent mammalian mammalian cell cell to atopluripotent a pluripotent mammalian mammalian cell, cell, and and

whereinthe wherein the mixture mixturedoes doesnot notcomprise comprisefeeder feedercells. cells.

[0004b]

[0004b] InIn a athird thirdaspect, aspect, the the present present invention invention provides provides a a composition comprising: composition comprising:

30 30 (a) (a) a Rho a Rho kinase(ROCK) kinase (ROCK) inhibitor; inhibitor;

2

(b) (b) an an ALK5 ALK5 inhibitor; inhibitor; 25 Jun 2025 2022204080 25 Jun 2025

(c) (c) a MAP/ERK a MAP/ERK kinase kinase (MEK) (MEK) inhibitor; inhibitor; andand

(d) (d) a moleculartether; a molecular tether; whereinthe wherein the ROCK ROCK inhibitor inhibitor is is presentininananamount present amount effectivetotoincrease effective increaseefficiency efficiency of of 55 reprogramming reprogramming a non-pluripotent a non-pluripotent mammalian mammalian cell to cell to a pluripotent a pluripotent mammalian mammalian cell, cell, and and wherein the molecular tether is capable of attaching a mammalian cell directly to a wherein the molecular tether is capable of attaching a mammalian cell directly to a

solid culturesurface. solid culture surface. 2022204080

[0004c]

[0004c] In In a a fourthaspect, fourth aspect,the the present present invention invention provides providesuse useof of aa composition compositioninininduction induction of of non-pluripotent non-pluripotent mammalian cellsinto mammalian cells intoaapluripotent pluripotent mammalian mammalian cell,wherein cell, wherein thethe

10 10 composition composition comprises: comprises:

(a) (a) a Rho a Rho kinase kinase (ROCK) (ROCK) inhibitor inhibitor

(b) (b) an an ALK5 ALK5 inhibitor;and inhibitor; and (c) (c) a MAP/ERK a MAP/ERK kinase kinase (MEK) (MEK) inhibitor; inhibitor;

whereinthe wherein the ROCK ROCK inhibitor inhibitor is is presentininananamount present amount effectivetotoincrease effective increaseefficiency efficiency of of 15 15 reprogramming reprogramming a non-pluripotent a non-pluripotent mammalian mammalian cell to cell to a pluripotent a pluripotent mammalian mammalian cell. cell.

[0004d] Thepresent

[0004d] The presentinvention inventionprovides provides formixtures for mixtures(e.g., (e.g., useful useful for for inducing iPSCs). InIn inducing iPSCs).

some embodiments, some embodiments, thethe mixture mixture comprises: comprises:

mammalian mammalian cells; cells;

20 a TGFß 20 a TGFβ receptor/ALK5 receptor/ALK5 inhibitor; inhibitor;

aa MEK inhibitor;and MEK inhibitor; and

aa Rho Rho GTPase/ROCK pathway GTPase/ROCK pathway inhibitor. inhibitor.

[0005]

[0005] In In some some embodiments, embodiments, at least at least 99% 99% of cells of the the cells areare non-pluripotent non-pluripotent cells.InInsome cells. some embodiments, embodiments, all all or essentially or essentially allthe all of of the cells cells are non-pluripotent are non-pluripotent cells. cells.

25 [0006] 25 [0006] In embodiments, In some some embodiments, theare the cells cells are cells. human human cells.

[0007]

[0007] InInsome someembodiments, embodiments,the theTGF TGFβ receptor/ALK5 receptor/ALK5 inhibitorisis SB431542. inhibitor SB431542.

[0008]

[0008] InInsome someembodiments, embodiments,the theMEK MEK inhibitoris inhibitor is PD0325901. PD0325901.

[0009]

[0009] In In some some embodiments, embodiments, the ROCK the ROCK inhibitor inhibitor is a compound is a compound having having the the formula: formula:

2a 2a

25 Jun 2025

N N L 1 L2 L¹-L² A A H IZ

B B N N

S S R¹1 R (I) (I)

ring ring AAis isaasubstituted substitutedororunsubstituted unsubstituted cycloalkyl, cycloalkyl, substituted substituted or unsubstituted or unsubstituted

heterocycloalkyl, substituted heterocycloalkyl, substituted or unsubstituted or unsubstituted aryl, aryl, or substituted or substituted or unsubstituted or unsubstituted heteroaryl; heteroaryl; 2022204080

ring ring BBis is aa substituted substitutedororunsubstituted unsubstituted heterocycloalkyl, or substituted or 2022204080

heterocycloalkyl, or substituted or

55 unsubstituted unsubstituted heteroaryl; heteroaryl;

L¹1 is L -C(O)-NR²-2-or is -C(O)-NR -C(O)-NR2-; or -C(O)-NR²-; L²2 is L is aa bond, bond,substituted substitutedor or unsubstituted unsubstituted alkylene alkylene or substituted or substituted or or unsubstituted heteroalkylene; unsubstituted heteroalkylene; and and R¹1 and R 2 independently hydrogen, substituted or unsubstituted alkyl, substituted or andR²Rare are independently hydrogen, substituted or unsubstituted alkyl, substituted or 10 10 unsubstituted unsubstituted heteroalkyl, heteroalkyl, substituted substituted oror unsubstitutedcycloalkyl, unsubstituted cycloalkyl,substituted substitutedor or unsubstituted unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl

[0010]

[0010] In In some some embodiments, embodiments, the ROCK the ROCK inhibitor inhibitor has thehas the formula: formula:

2b 2b

N L² N-2 IZ ZI N (R³) HH H Rz N N Z (R) (R4 y X S S R( R¹ (II) y

wherein,y yisis an ;R 3 wherein, aninteger integerfrom 0 to 3; Z is an integer from 0 to 5; X is -N=, -CH= or -CR; R³, from 0 to 3; z is an integer from 0 to 5; X is -N=, -CH= or -C

, 2022204080

R4and R and RR 5are are independently independentlyCN, CN, S(O)nR', S(O)nR, NRR, C(O)R, NR'R', 9, NR C(O)RNR¹-C(O)R¹, °-C(O)R", NR¹²-C(O)-OR¹³, NR -C(O)-ORD,-C(O)NR¹R¹, -NR¹S(O)2R¹, -C(O)NR 14R -OR¹, -S(O)2NR¹, 5, -NR 16S(O)2R7, substituted -OR", -S(O)2NR, or substituted or 55 unsubstituted unsubstituted alkyl, alkyl, substituted substituted or unsubstituted or unsubstituted heteroalkyl, heteroalkyl, substituted substituted or unsubstituted or unsubstituted

cycloalkyl, substituted cycloalkyl, substitutedororunsubstituted unsubstitutedheterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl,aryl, or or substituted or substituted or unsubstituted unsubstitutedheteroaryl, heteroaryl,wherein wherein n an n is is an integer integer fromfrom 0 to 02, towherein 2, wherein if Z if is z is greater than greater than 1,1, two twoR³R3moieties moietiesareareoptionally optionally joined joined together together to form to form a substituted or a substituted or unsubstitutedcycloalkyl, unsubstituted cycloalkyl,substituted substitutedor or unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or or 7 76 8 9 10 1 12 10 10 unsubstituted aryl,aryl, unsubstituted or substituted or unsubstituted or substituted heteroaryl; or unsubstituted and R,and heteroaryl; R6R, R, R, , RR¹, , RR¹¹, , R R¹², , R1 , R", R R¹³, R¹, R 1, R 14,R¹, R R¹, R¹,R17, , R 1, R¹ and R"R¹and areR'9 independently hydrogen, substituted or unsubstituted are independently hydrogen, substituted or unsubstituted alkyl, substituted alkyl, or unsubstituted substituted or unsubstitutedheteroalkyl, heteroalkyl,unsubstituted unsubstituted cycloalkyl, cycloalkyl, substituted substituted or or unsubstituted heterocycloalkyl,substituted unsubstituted heterocycloalkyl, substituted or unsubstituted or unsubstituted aryl, aryl, or substituted or substituted or or

unsubstitutedheteroaryl. unsubstituted heteroaryl.

15 15 [00111

[0011] In some In some embodiments, embodiments, the inhibitor the ROCK ROCK inhibitor hasformula: has the the formula:

0 O N N ZI H NN N H 1~H IZ N H

N S N N O N H NSD N ZI H ZI N H H H N O S

0 O N N H H ZI IZ N N H H N N

N N SS

0 O N NN IZ ZI N NH H H N N

S S ,or , or

3

0 O N N ZI H ZI N H H H 2022204080 10 Jun N\ N N N

N N S SOCH OCH. 3

.

[00121 InInsome

[0012] someembodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor is is

0 O NN N IZ

H ZI N H N H N H N N N N S S

[0013] In some

[0013] In some embodiments, embodiments, the concentration the concentration of the inhibitors of the inhibitors is sufficient is sufficient to to improve improve 5 by at 5 by least at least 10% 10% the efficiency the efficiency of induction of induction of non-pluripotent of non-pluripotent cells incells in the mixture the mixture into into inducedpluripotent induced pluripotentstem stem cellswhen cells when the the mixture mixture is submitted is submitted to conditions to conditions sufficient sufficient to to induceconversion induce conversionof of thethe cellsinto cells intoinduced induced pluripotent pluripotent stemstem cells. cells.

[0014] InInsome

[0014] someembodiments, embodiments, thethe mixture mixture furthercomprises further comprisesa aGSK3 GSK3 inhibitorand/or inhibitor and/or HDAC inhibitor. HDAC inhibitor.

10 [0015] 10 [0015] In some In some embodiments, embodiments, the polypeptides the polypeptides are selected are selected fromfrom Oct-3/4, Oct-3/4, Sox2, Sox2, KLF4KLF4 and and c-Myc. InIn some c-Myc. someembodiments, embodiments,thethecells cells are are selected selected from from human cell, non-human human cell, animal non-human animal

cells, cells, mouse cells, non-human mouse cells, non-human primates, primates, or other or other animal animal cells.cells.

[0016] The The

[0016] present present invention invention also provides methodsmethods also provides of inducing of inducing non-pluripotent non-pluripotent

mammalian cells mammalian cells intointo induced induced pluripotent pluripotent stem stem cells.cells. In embodiments, In some some embodiments, the methodthe method

15 comprises 15 comprises

contacting non-pluripotent contacting non-pluripotent cellswith: cells with:

a TGFB receptor/ALK5inhibitor; TGFß receptor/ALK5 inhibitor;

aa MEK inhibitor; and MEK inhibitor; and

aa ROCK inhibitor, ROCK inhibitor,

20 underunder 20 conditions conditions sufficient sufficient to induce to induce at some at least least cells sometocells to become become pluripotent pluripotent stem stem cells. cells.

[0017] InInsome

[0017] someembodiments, embodiments, thethe conditionscomprise conditions comprise introducingatatleast introducing least one one exogenous exogenous

transcription factor transcription factor into into the the non-pluripotent non-pluripotentcells. cells.InInsome some embodiments, embodiments, the at the at least least one one exogenous transcription exogenous transcription factor factor is is an an OctOct polypeptide polypeptide andcells and the the cells are further are further contacted contacted with awith a

histone deacetylase(HDAC) histone deacetylase (HDAC) inhibitor. inhibitor.

4

[00181 In some

[0018] In some embodiments, embodiments, the transcription the transcription factor factor is is selected selected from thefrom groupthe group consisting consisting

of an of Octpolypeptide, an Oct polypeptide,a aKlfKlf polypeptide, polypeptide, a Myc a Myc polypeptide, polypeptide, and a and a Sox polypeptide. Sox polypeptide.

[00191 In some

[0019] In some embodiments, embodiments, the comprises the method method comprises introducingintroducing at three at least two, least two, three or four or four exogenous exogenous transcription transcription factor factor into into thethe non-pluripotent non-pluripotent cells, cells, wherein wherein the transcription the transcription factors factors

5 are are 5 selected selected fromfrom the group the group consisting consisting of an of an Oct Oct polypeptide, polypeptide, a Klf polypeptide, a Klf polypeptide, a Myc a Myc

2022204080 polypeptide,and polypeptide, anda aSoxSox polypeptide. polypeptide. In some In some embodiments, embodiments, the polypeptides the polypeptides are are selected selected from Oct-3/4, from Oct-3/4, Sox2, Sox2, KLF4 andc-Myc. KLF4 and c-Myc.InInsome some embodiments, embodiments, thethe cellsare cells areselected selected from from humancell, human cell,non-human non-human animal animal cells,cells, mousemouse cells, cells, non-human non-human primates,primates, or other or other animal animal cells. cells.

[00201 In some

[0020] In some embodiments, embodiments, the atone the at least least one transcription transcription factor factor is is introduced introduced by by 10 10 introducing introducing a polynucleotide a polynucleotide into into the the non-pluripotent non-pluripotent cells, wherein cells, wherein the polynucleotide the polynucleotide

encodesthe encodes theatatleast least one oneexogenous exogenous transcription transcription factor, factor, thereby thereby expressing expressing the transcription the transcription

factor(s) in the cells. factor(s) in the cells.

[00211 In some

[0021] In some embodiments, embodiments, the atone the at least least one transcription transcription factor factor is is introduced introduced by by contactingananexogenous contacting exogenous polypeptide polypeptide to non-pluripotent to the the non-pluripotent cells, cells, wherein wherein the polypeptide the polypeptide

15 15 comprises comprises the amino the amino acid sequence acid sequence of the transcription of the transcription factor, the factor, wherein wherein the introduction introduction is is performedunder performed under conditions conditions to introduce to introduce the polypeptide the polypeptide intocells. into the the cells. In In some some embodiments, embodiments, the the polypeptide polypeptide comprises comprises an acid an amino amino acid sequence sequence that transport that enhances enhances transport across cell across cell membranes. membranes.

[0022] InInsome

[0022] some embodiments, embodiments, thethe cellsare cells arehuman humancells. cells.

20 [0023] 20 [00231 In some In some embodiments, embodiments, the receptor/ALK5 the TGFß TGFP receptor/ALK5 inhibitor inhibitor is SB431542. is SB431542.

[00241 InInsome

[0024] some embodiments, embodiments, thethe MEKMEK inhibitor inhibitor is PD0325901 is PD0325901

[0025] InInsome

[0025] some embodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor is ais compound a compound having having the the formula: formula:

N ' L¹-L² L'L2 A A H ZI H B B N N

S S R¹1() (I)

ring AA isis aa substituted ring substituted or or unsubstituted unsubstitutedcycloalkyl, cycloalkyl,substituted substituted or or unsubstituted unsubstituted

25 heterocycloalkyl, 25 heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl, aryl, or or substituted substituted or unsubstituted or unsubstituted heteroaryl; heteroaryl;

ring BB is ring is aa substituted or unsubstituted substituted or unsubstitutedheterocycloalkyl, heterocycloalkyl, or or substituted substituted or or unsubstitutedheteroaryl; unsubstituted heteroaryl; L¹ is-C(O)-NR -C(O)-NR²- 2 2 or2_-C(O)-NR²-; L is - or -C(O)-NR2_

5

L² L2 is is aa bond, bond, substituted substituted or unsubstituted alkylene or substituted or or unsubstituted alkylene or substituted or unsubstitutedheteroalkylene; unsubstituted heteroalkylene;andand R¹ and R1 andR²R2are areindependently independentlyhydrogen, substituted hydrogen, or unsubstituted alkyl, substituted or substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, unsubstituted heteroalkyl,substituted substitutedor orunsubstituted unsubstituted cycloalkyl, cycloalkyl, substituted substituted or unsubstituted or unsubstituted

5 heterocycloalkyl, 5 heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl, aryl, or or substituted substituted or unsubstituted or unsubstituted heteroaryl. heteroaryl.

2022204080 100261 InInsome

[0026] some thethe embodiments, embodiments, ROCK ROCK inhibitor thethe has has inhibitor formula: formula:

0 O N L² ZI NNH / IZ N L2 H (R³) N N H R3) N N I \ z Z (R) (R4 y S S R(z R¹ X (II) y

wherein,y yisis ananinteger wherein, integerfrom 3; z is an integer from 0 to 5; X is -N=, -CH= or -CR=;R3 from0 0toto3; Z is an integer from 0 to 5; X is -N=, -CH= or -CR=; R³,

, R 4and R and RR are are independently independently CN, CN, S(O)nR6 , NRR, S(O)nR, NRR', C(O)R, NR¹-C(O)R¹, C(O)R 9 , NR'°-C(O)R'

, 10 , -C(O)NR1 4R1, NR 1-C(O)-OR13-C(O)NR¹R¹, 10 NR¹²-C(O)-OR¹³, -NR16S(O)2R", -NR¹S(O)2R¹, -OR'", -OR¹, -S(O)2NR, -S(O)2NR¹, substitutedoror substituted

unsubstitutedalkyl, unsubstituted alkyl,substituted substitutedororunsubstituted unsubstituted heteroalkyl, heteroalkyl, substituted substituted or unsubstituted or unsubstituted

cycloalkyl, substituted cycloalkyl, substitutedororunsubstituted unsubstitutedheterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl,aryl, or or substituted or substituted or unsubstituted unsubstitutedheteroaryl, heteroaryl,wherein wherein n an n is is an integer integer from from 0 to 02, towherein 2, wherein if Z if is z is greater greater than than 1,1, two twoR³R3moieties moietiesareare optionally joined optionally together joined to form together a substituted to form or a substituted or 15 unsubstituted 15 unsubstituted cycloalkyl, cycloalkyl, substituted substituted or unsubstituted or unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or or unsubstituted 8 R¹¹, 9 108 12 unsubstitutedaryl, aryl, or or substituted substitutedororunsubstituted unsubstitutedheteroaryl; andand heteroaryl; R, R, R ,R,R R,, R, R¹, R¹², R9, R1°, R1, R 13 R¹³, 14 15R¹, 16 17 and189 R , R¹, R ,R¹, R", R¹, R¹ R¹ are independently hydrogen, substituted or unsubstituted R1 , R , R" and R" are independently hydrogen, substituted or unsubstituted alkyl, substituted alkyl, substituted or or unsubstituted unsubstitutedheteroalkyl, heteroalkyl,unsubstituted unsubstituted cycloalkyl, cycloalkyl, substituted substituted or or unsubstitutedheterocycloalkyl, unsubstituted heterocycloalkyl, substituted substituted or or unsubstituted unsubstituted aryl, aryl, or substituted or substituted or or 20 unsubstituted 20 unsubstituted heteroaryl. heteroaryl.

[0027] InInsome

[0027] some embodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor has has the the formula: formula:

0 O N N N IZ N H ZI H H H N

N S

0 0 N N N IZ

H6 IZ N H H N S S

O N N IZ H ZI N H H H N N\ N N N N S

0 O N ZI N ZI N N H H 2022204080 N N

S S or , or

0 O N ZI N N N H H H H N N

N N S SOCH OCH. 3

.

[0028] InInsome

[0028] someembodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor is is 0 O N NNIZ ZI N N H H N N N

5 5 N N S S

[00291 In some

[0029] In some embodiments, embodiments, the concentration the concentration of the inhibitors of the inhibitors is sufficient is sufficient to improve to improve by by at at least least 10% theefficiency 10% the efficiencyofofinduction inductionof of non-pluripotent non-pluripotent cells cells in the in the mixture mixture into into induced induced

pluripotent stemcells, pluripotent stem cells, when when the the mixture mixture is subjected is subjected to conditions to conditions sufficient sufficient to induce to induce

conversion conversion ofof thecells the cellsinto intoinduced induced pluripotent pluripotent stem stem cells. cells.

10 [0030] 10 [0030] In some In some embodiments, embodiments, the mixture the mixture further further comprises comprises a GSK3 a GSK3 inhibitor. inhibitor.

[0031] The The

[0031] present present invention invention also provides also provides forfor for kits kitsinducing for inducing pluripotency pluripotency in non- in non pluripotent mammalian pluripotent mammalian cells. cells. In some In some embodiments, embodiments, the kit comprises, the kit comprises,

a TGFB a receptor/ALK5inhibitor; TGFß receptor/ALK5 inhibitor;

a MEK a inhibitor; and MEK inhibitor; and

15 a ROCK 15 a ROCK inhibitor. inhibitor.

[00321 InInsome

[0032] some embodiments, embodiments, thethe TGFP TGFß receptor/ALK5 receptor/ALK5 inhibitor inhibitor is SB431542. is SB431542.

[0033] InInsome

[0033] some embodiments, embodiments, thethe MEKMEK inhibitor inhibitor is PD0325901. is PD0325901.

[0034] InInsome

[0034] some embodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor is ais compound a compound having having the the formula: formula:

N N L¹-L²2 A A H ZI H B B N N S S R( R¹ (I)

ring AA isis aa substituted ring or unsubstituted substituted or unsubstitutedcycloalkyl, substituted cycloalkyl,substituted or or unsubstituted unsubstituted

2022204080

heterocycloalkyl,substituted heterocycloalkyl, substitutedororunsubstituted unsubstituted aryl, aryl, or or substituted substituted or or unsubstituted unsubstituted heteroaryl; heteroaryl;

ring BB is ring is aa substituted or unsubstituted substituted or unsubstitutedheterocycloalkyl, heterocycloalkyl,or or substituted substituted or or 55 unsubstituted unsubstitutedheteroaryl; heteroaryl; 2 or -C(O)-NR²-; Ll is L¹ is-C(O)-NR -C(O)-NR²-- or -C(O)-NR L² L2 is is aa bond, bond, substituted substituted ororunsubstituted unsubstitutedalkylene or or alkylene substituted or substituted or unsubstitutedheteroalkylene; unsubstituted heteroalkylene;andand R¹ R1 and andR²R2are areindependently hydrogen, independently substituted hydrogen, or unsubstituted substituted alkyl, alkyl, or unsubstituted substituted or substituted or 10 unsubstituted 10 unsubstituted heteroalkyl, heteroalkyl, substituted substituted or unsubstituted or unsubstituted cycloalkyl, cycloalkyl, substituted substituted or unsubstituted or unsubstituted

heterocycloalkyl,substituted heterocycloalkyl, substitutedororunsubstituted unsubstituted aryl, aryl, or or substituted substituted or or unsubstituted unsubstituted heteroaryl heteroaryl

[00351 InInsome

[0035] someembodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor has has the the formula: formula:

0 O N N L2 L² IZ ZI N (R³) N H H R3) N (R4) Z (R4 X S S R1Iz R¹ X (II) y

wherein, wherein,y is an integer from 0 to 3; Z is an integer from 0 to 5; X is -N=, -CH= or -CR=; R³, y is an integer from 0 to 3; z is an integer from 0 to 5; X is -N, -CH= or -CR= R, 15 R R4 15 andandR Rare are independently independently CN, CN, S(O)nR, S(O)nR', NRR, C(O)R9, NR'R', C(O)R, NR'°-C(O)R", NR¹-C(O)R¹, -C(O)NR 1-NR¹S(O)2R¹, NR -C(O)-OR", -C(O)NR NR¹²-C(O)-OR¹³, -OR¹,7, -OR", 4R", -NR 16S(O)2R -S(O)2NR, -S(O)2NR¹, substituted substituted or or unsubstitutedalkyl, unsubstituted alkyl,substituted substitutedororunsubstituted unsubstituted heteroalkyl, heteroalkyl, substituted substituted or unsubstituted or unsubstituted

cycloalkyl, substituted cycloalkyl, substituted ororunsubstituted unsubstitutedheterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl, aryl, or or substituted or unsubstituted substituted or heteroaryl,wherein unsubstitutedheteroaryl, wherein n isn an is an fromfrom integer integer 0 to 02,towherein 2, wherein if z is if Z is

20 20 greater thanthan greater 1, two 1, R³ R 3 moieties twomoieties are optionally joined joined are optionally togethertogether to form to form a substituted a substituted or or unsubstitutedcycloalkyl, unsubstituted cycloalkyl,substituted substituted or or unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or or 8 R¹¹, 76 R¹, unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R, R,6 R, R, 9 10 R¹², 11 12 unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R , R , R , R , R , R , R 13 R¹, R¹³, 14 R¹,15R¹, 16 17 and189 R¹, R¹ R¹ are independently hydrogen, substituted or unsubstituted RD, R , R , R , R , R" and R'9 are independently hydrogen, substituted or unsubstituted alkyl, alkyl, substituted or unsubstituted substituted or heteroalkyl,unsubstituted unsubstitutedheteroalkyl, unsubstituted cycloalkyl, cycloalkyl, substituted substituted or or

25 unsubstituted 25 unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted aryl, or aryl, or unsubstituted or substituted substituted or or unsubstituted heteroaryl. unsubstituted heteroaryl.

[00361 InInsome

[0036] some embodiments, embodiments, thethe ROCK ROCK inhibitor inhibitor has has the the formula: formula:

0 O N N NIZ N H ZI H H H N

N S S N 0 O 2022204080 N N IZ

H ZI N N H N H N H< S S ,

0 O N IZ ZI N H H H N N N

N N S S

0 O N NNIZ ZI H N H H N N

5 5 d sior S , or

0 O N N IZ ZI N N H N_ H N N

N N S S 0CH3 OCH. .

[00371 InInsome

[0037] embodiments, some embodiments, thethe ROCK ROCK inhibitor inhibitor is is

0 O N NNIZ HH ZI N H H N N N N -- I

N S

[00381 In some

[0038] In some embodiments, embodiments, the kit the kit further further comprises comprises aGSK3 and/or a GSK3 inhibitor inhibitor and/or a histone ahistone 10 deacetylase 10 deacetylase (HDAC (HDAC) inhibitor. inhibitor.

[0039] Other

[0039] Other embodiments embodiments will be will clearbefrom clear from the the remainder remainder of this disclosure. of this disclosure.

DEFINITIONS DEFINITIONS

[0040] AnAn

[0040] "Oct "Oct polypeptide"refers polypeptide" referstoto any any of of the the naturally-occurring naturally-occurringmembers members of of Octamer Octamer

family ofoftranscription family transcription factors, factors, oror variants variantsthereof thereofthat thatmaintain maintain transcription transcription factor factor activity, activity,

15 similar 15 similar (within (within at least at least 50%,50%, 80%, 80%, or 90% or 90% activity) activity) comparedcompared to therelated to the closest closestnaturally related naturally

occurring family member, occurring family polypeptides comprising or polypeptides member, or at least comprising at leastthe the DNA-binding domain DNA-binding domain of of

the naturally the occurringfamily naturally occurring familymember, member, andfurther and can can further comprise comprise a transcriptional a transcriptional activation activation

Exemplary domain.Exemplary domain. Oct polypeptides Oct polypeptides include, include, Oct-1, Oct-3/4, Oct-1, Oct-2, Oct-2, Oct-3/4, Oct-6,Oct-8, Oct-6, Oct-7, Oct-7, Oct-8, Oct-9, andOct-11. Oct-9, and Oct-11.e.g. e.g.Oct3/4 Oct3/4 to to (referred (referred herein herein as "Oct4") as "Oct4") contains contains the POU POU domain, the domain, a 150 a 150 5 amino 5 amino acidacid sequence sequence conserved conserved among among Pit-, Pit-l, Oct-1, Oct-1, Oct-2,and Oct-2, anduric-86. uric-86. See, See, Ryan, Ryan,A.K. A.K.&

& Rosenfeld, M.G. Rosenfeld, Dev.11, GenesDev. M.G. Genes 11, 1207-1225 (1997). InInsome 1207-1225(1997). embodiments, someembodiments, variants haveatat variantshave 2022204080

least 85%, least 90%, 85%, 90%, or or 95% 95% amino amino acid sequence identityidentity acid sequence across their theirsequence acrosswhole whole sequence compared compared to aa naturally to occurringOct naturally occurring Octpolypeptide polypeptide family family member member such assuch as tolisted to those those above above listed or such or such as as listed listedinin Genbank accessionnumber Genbankaccession number NP_002692.2 (human NP_002692.2 (human Oct4)ororNP_038661.1 Oct4) NP_038661.1 (mouse (mouse

10 10 Oct4). Oct4). Oct polypeptides Oct polypeptides (e.g., (e.g., Oct3/4) can be can Oct3/4) from be frommouse, human, human, rat,mouse, bovine,rat, bovine, porcine, or porcine, or other animals.Generally, other animals. thethe Generally, same same species species of protein of protein will will be used be used withspecies with the the species of cells of cells

being manipulated. being manipulated.

[00411 A "Klf

[0041] A "Klf polypeptide" polypeptide" refersrefers to anytoofany theof the naturally-occurring naturally-occurring members members of the of the family family of Krüppel-like of Kruppel-likefactors (Klfs),zinc-finger factors(Klfs), proteins zinc-fingerproteins thatcontain that contain amino amino acid acid sequences sequences similar similar

15 15 to those to those of the of the Drosophila Drosophila pattern pattern embryonic embryonic regulator regulator Krüppel, KrUppel, or of or variants variants of the naturally the naturally-

occurringmembers occurring membersthatthat maintain maintain transcription transcription factor factor activity activity similar similar (within (within at least at least 50%, 50%

, 80%,oror90% 80%, 90% activity) activity) compared compared to closest to the the closest related related naturally naturally occurring family family occurring member,member, or or polypeptidescomprising polypeptides comprising at least at least thethe DNA-binding DNA-binding domain domain of the naturally of the naturally occurringoccurring family family member, member, andand cancan further further comprise comprise a transcriptional a transcriptional activation activation domain. domain. See,D.T., See, Dang, Dang, D.T., 20 Pevsner, 20 J. J. Pevsner, && Yang, Yang, V.W.. V.W.. Cell Cell Biol.32, Biol. 32,1103-1121 (2000).Exemplary 1103-1121 (2000). Exemplary Klf Kf family family members members

include, Klfl, include, Klfl, Klf2, Klf2, Klf3, Klf3,Klf-4, Klf-4,Klf5, Klf5,Klf6, Klf6,Klf7, Klf7, Klf8, Klf8, Klf9, Klf9, Kfl0, Klf10, Klf11, Klfl1, Klfl2, Klf12, KlfI Klf13, 3, Klfl4, Klf15, Klf14, KlfI6,andand Klfl5,Klf16, Klfl7. Klf17. Klf2 Klf2 and Klf-4 and Klf-4 were tofound were found to be factors be factors capable capable of of generatingiPS generating cellsininmice, iPScells mice,andand related related genes genes KlfI Klfl Klf5 Klf5 and and didwell, did as as well, although although with with reducedefficiency. reduced See, efficiency.See, Nakagawa, Nakagawa, et al., et al., Nature Nature Biotechnology Biotechnology 26:101 -26:101 - 106In(2007). 106 (2007). In 25 somesome 25 embodiments, embodiments, havehave variants variants at least at least 85%, 85%, 90%, 90%, or 95% or 95% amino acidacid amino sequence sequence identity identity

across their across their whole sequence wholesequence compared compared to a naturally to a naturally occurring occurring Klf polypeptide Klf polypeptide family family member member such such as those as to to those listed listed above above or such or such as listed as listed in Genbank in Genbank accession accession number number CAX16088 CAX16088 (mouse (mouse Klf4) Klf4) or or CAX14962 CAX14962 (human (human Klf4).Klf4). Klf polypeptides (e.g.,(e.g., Klf polypeptides Klfl,KifKlf4, Klf4,

Klf5)can and Klf5) and canbebefrom from human, human, mouse, mouse, rat, bovine, rat, bovine, porcine, porcine, or animals. or other other animals. Generally, Generally, the the 30 samesame 30 species species of protein of protein will will be usedthe bewith used with the species species of cellsof cellsmanipulated. being being manipulated. To the To the extent aa Klf extent polypeptideisisdescribed Klf polypeptide described herein, herein, it it can be be can replaced replaced withwith an estrogen-related an estrogen-related

beta(Essrb) receptor beta receptor (Essrb)polypeptide. polypeptide. Thus, Thus, it intended it is is intended thatthat for for eacheach Klf Klf polypeptide polypeptide

embodiment embodiment described described herein, herein, a corresponding a corresponding embodiment embodiment using using Essrb Essrb in the thea place placeinof Klf4 of a Klf4 polypeptideisisequally polypeptide equallydescribed. described.

10

[0042] A A

[0042] "Myc "Myc polypeptide" polypeptide" anyofofthe refersany refers naturally-occurring members the naturally-occurring of the members of the Myc Myc

family (see, family e.g., Adhikary, (see, e.g., S. Eilers, Adhikary, S.& M. M. & Eilers, Nat. Rev.Rev. Nat. Mol.Mol. Biol.Biol. Cell Cell 6:635-645 6:635-645 (2005)), (2005)), or or variants thereofthat variants thereof that maintain maintaintranscription transcriptionfactor factoractivity activitysimilar similar(within (within at at least50%, least 50%, 80%, 80%,

or 90% or 90%activity) activity)compared compared to the to the closest closest related related naturally naturally occurring occurring family family member, member, or or 5 polypeptides 5 polypeptides at at comprising comprising leastthe least the DNA-binding domain DNA-bindingdomain thethe of of naturally occurring naturally family occurring family andcan member,and member, further comprise canfurther comprise aa transcriptional transcriptional activation domain. activation domain. ExemplaryMyc Exemplary Myc 2022204080

polypeptides include,e.g., polypeptides include, e.g.,c-Myc, N-Myc and N-Myc c-Myc, and L-Myc. L-Myc. In someembodiments, In some variantshave embodiments,variants have least 85%, at least at 90%,oror95%95% 85%, 90%, amino amino acid sequence acid sequence identity identity across across their sequence their whole whole sequence compared compared to to a naturally a naturally Myc Myc occurring occurring polypeptide family family polypeptide member, member, such as tosuch aslisted those to those listed 10 10 above above or such or such as as listedinin Genbank listed accessionnumber Genbankaccession CAA25015 numberCAA25015 (human Myc).Myc). (human Myc Myc polypeptides(e.g., polypeptides c-Myc)cancan (e.g.,c-Myc) be be from from human, human, mouse,mouse, rat, bovine, rat, bovine, porcine, or otheroranimals. porcine, other animals. thesame Generally,the Generally, samespecies of of species protein protein will will be used be used withwith the species the species of cells beingbeing of cells manipulated. manipulated.

[0043] A A

[0043] "Sox "Sox polypeptide" polypeptide" refers any of referstoto any the naturally-occurring of the members of naturally-occurringmembers theSRY of the SRY-

related 15 related 15 HMG-box HMG-box (Sox) transcription factors, factors, (Sox) transcription characterized characterized by the ofpresence by the presence of the the high- high mobilitygroup mobility (HMG) group(HMG) domain, domain, or variants or variants thereofthereof that maintain that maintain transcription transcription factor activity factor activity

(within atat least similar (within similar least 50%, 50%,80%, 80%,or or 90%90% activity) activity) compared to the to compared the closest closest related related naturally naturally

occurring family occurring family member, polypeptides comprising orpolypeptides member, or at least comprising at least the DNA-binding domain theDNA-binding of domain of

the naturally the naturally occurring familymember, occurringfamily member, andfurther and can can further comprise comprise a transcriptional a transcriptional activation activation

domain. 20 domain. 20 e.g., Dang, See, Dang, See, e.g., D.T., D.T., et al.,et al., J. Int. J. Biochem. Int.Biochem. Cell Biol. Biol. 32:1103-1121 Cell32:1103-1121 (2000). (2000). Exemplary Exemplary SoxSox polypeptides polypeptides include, include, e.g., e.g., Sox1,Soxi, Sox-2,Sox-2, Sox3,Sox5, Sox3, Sox4, Sox4,Sox6, Sox5, Sox6, Sox7, Sox7, Sox8, Sox9, Sox8, Sox1, Sox11, Sox9, Sox10, SoxI1, Sox12, Sox13,Sox14, Sox12,Sox13, Sox15,Sox17, Sox14,Sox15, Sox17,Sox18, Sox-21,andandSox30. Sox8,Sox-21, Sox30. SoxIhas Sox1 hasbeen beenshown shown to yield to yield iPS cells withwith iPS cells a similar a similar efficiency efficiency as Sox2, as Sox2, and Sox3, and genes genes Sox3, andSox18 Sox15,and Sox15, Sox18 havehave been been also also shown shown to generate to generate iPSalthough iPS cells, cells, although with less with somewhat somewhat less 25 efficiency 25 efficiency thanSox2. than Sox2.See, See,Nakagawa, Nakagawa, et al.,Nature et al., Biotechnology NatureBiotechnology 26:101 - 106 26:101106 (2007). (2007). In In some embodiments, some embodiments,variants have at variants have at least 85%, 90%, least 85%, 90%, or or 95% 95% amino acid sequence aminoacid sequenceidentity identity across their across their whole wholesequence sequence compared compared to a naturally to a naturally occurring occurring Sox polypeptide Sox polypeptide family family member member such such as those as to to those listed listed above above or such or such as listed as listed in Genbank in Genbank accession accession number number CAA83435 CAA83435 (human (human Sox Sox Sox2). Sox2). polypeptides polypeptides (e.g., (e.g., Sox1, Sox1, Sox2, Sox2, Sox3, Sox3, Sox15, Sox15, or or Sox18) Sox18) cancan

30 be from 30 be from mouse, mouse, human, human, rat, bovine, porcine,porcine, rat, bovine, or other or animals. Generally, Generally, other animals. the same the same species of species of protein will protein be used will be usedwith withthe thespecies of of species cellsbeing cells being manipulated. manipulated.

[00441 "H3K9"

[0044] "H3K9" to to refers refers histoneH3H3lysine histone lysine 9. 9. H3K9 H3K9 associatedwith modificationsassociated modifications withgene gene activity include activity H3K9 include H3K9 acetylation acetylation and and H3K9H3K9 modifications modifications associated associated with heterochromatin, with heterochromatin,

11

include H3K9 include H3K9 di-methylation di-methylation or tri-methylation. or tri-methylation. See, e.g., See, e.g., Kubicek, Kubicek, et al.,etMol. al., Cell473-481 Mol. Cell473-481 (2007). (2007).

[0045] TheThe

[0045] termterm "pluripotent" "pluripotent" or "pluripotency" or "pluripotency" refers refers to with to cells cellsthe with the ability ability to rise to give give rise to progeny to progenycells cellsthat thatcan canundergo undergo differentiation, differentiation, under under the the appropriate appropriate conditions, conditions, into into cell cell 55 types types thatthat collectively collectively demonstrate demonstrate characteristics characteristics associated associated withlineages with cell cell lineages from allfrom all of the of the

2022204080 three germinal three germinallayers layers(endoderm, (endoderm, mesoderm, mesoderm, and ectoderm). and ectoderm). Pluripotent Pluripotent stem cellsstem can cells can contribute to contribute to all all embryonic derived embryonic derived tissues tissues of of a prenatal, a prenatal, postnatal postnatal or adult or adult animal. animal. A A standardart-accepted standard art-acceptedtest, test, such suchasasthe theability abilitytoto form forma ateratoma teratomain in 8-12 8-12 week week old SCID old SCID mice, mice, can be can be used usedtotoestablish establishthe thepluripotency pluripotencyof of a cellpopulation, a cell population, however however identification identification of of 10 various 10 various pluripotent pluripotent stem stem cell characteristics cell characteristics can bealso can also betoused used to detect detect pluripotent pluripotent cells. cells.

[00461 "Pluripotent

[0046] "Pluripotent stemstem cell cell characteristics" characteristics" referrefer to characteristics to characteristics of a of a cell cell thatthat

distinguish pluripotent distinguish pluripotentstem stemcells cellsfrom from other other cells.TheThe cells. ability ability to give to give riserise to to progeny progeny that that can can undergodifferentiation, undergo differentiation,under underthethe appropriate appropriate conditions, conditions, intointo cellcell types types thatthat collectively collectively

demonstratecharacteristics demonstrate characteristicsassociated associated with with cellcell lineages lineages fromfrom allthe all of of the three three germinal germinal layerslayers

15 (endoderm, 15 (endoderm, mesoderm, mesoderm, and ectoderm) and ectoderm) is a pluripotent is a pluripotent stem cell characteristic. stem cell characteristic. Expression Expression or or non-expressionof of non-expression certaincombinations certain combinations of molecular of molecular markers markers are alsoare also pluripotent pluripotent stem stem cell cell characteristics. For characteristics. Forexample, example, human human pluripotent pluripotent stem cells stem cells express express at some, at least least some, and in and some in some embodiments, embodiments, allall of of themarkers the markers fromfrom the following the following non-limiting non-limiting list: SSEA-3, list: SSEA-3, SSEA-4, SSEA-4, TRA-1-60,TRA-1-81, TRA-1-60, TRA-1-81,TRA-2-49/6E, TRA-2-49/6E, ALP,ALP, Sox2,Sox2, E-cadherin, E-cadherin, UTF-1, UTF-1, Oct4,Oct4, Rex1,Rexi, and and 20 Nanog. 20 Nanog. Cell morphologies Cell morphologies associated associated with pluripotent with pluripotent stem cells stem cellspluripotent are also are also pluripotent stem stem cell characteristics. cell characteristics.

[00471 As used

[0047] As used herein, herein, "non-pluripotent "non-pluripotent cells" cells" refer refer to mammalian to mammalian cells cells that are that not are not pluripotent cells. pluripotent cells. Examples Examples of of such such cells cells include include differentiated differentiated cellscells as well as well as progenitor as progenitor

cells. Examples cells. Examples of of differentiated differentiated cells cells include, include, butbut areare notnot limited limited to, to, cells cells from from a tissue a tissue

25 selected 25 selected from from bone marrow, bone marrow, skin, skeletal skin, skeletal muscle, muscle, fatand fat tissue tissue and peripheral peripheral blood. blood. Exemplary Exemplary cell types cell include, but types include, but are are not not limited limitedto, to, fibroblasts, fibroblasts, hepatocytes, hepatocytes,myoblasts, myoblasts, neurons, neurons,

osteoblasts, osteoclasts, osteoblasts, osteoclasts, and andT-cells. T-cells.

[0048] In some

[0048] In some embodiments embodiments where anwhere an individual individual is to bewith is to be treated treated with the the resulting resulting pluripotent cells, pluripotent cells, the individual's own the individual's ownnon-pluripotent non-pluripotent cells cells areare used used to generate to generate pluripotent pluripotent

30 cellscells 30 according according to methods to the the methods of the of the invention. invention.

[0049] Cells

[0049] Cellscan canbebefrom, from,e.g., e.g., humans or non-human humans or mammals. non-human mammals. Exemplary Exemplary non-human non-human

mammals mammals include, include, but but are are not not limited limited to, mice, to, mice, rats,rats, cats, cats, dogs, dogs, rabbits, rabbits, guinea guinea pigs,pigs, hamsters, hamsters,

12

pigs, horses, sheep, pigs, sheep, horses, bovines, andnon-human bovines,and non-human primates (e.g.,(e.g., primates chimpanzees, chimpanzees, macaques, macaques, and and apes). apes).

[0050]

[00501 A "recombinant" polynucleotide A "recombinant" is a polynucleotide is a polynucleotide polynucleotide thatinisits that is not notnative in its state, native state, e.g., the e.g., the polynucleotide comprises polynucleotide comprises a nucleotide a nucleotide sequence sequence not found not found in nature, in nature, or the or the 5 polynucleotide 5 is in isa in polynucleotide otherother a context context than in than that that in which which it is naturally it is naturally e.g., e.g., found,found, separated separated

2022204080 from nucleotidesequences from nucleotide withwith sequences which which it typically it typically in proximity is inisproximity in nature, in nature, or adjacent or adjacent (or (or with)nucleotide contiguouswith) contiguous nucleotide with with sequences sequences whichwhich it typically is notisin it typically not in proximity. proximity. For For thesequence example,the example, sequence at issue at issue cancan be cloned into into be cloned a vector, a vector, or otherwise or otherwise recombined with onewith recombined one or moreadditional or more additionalnucleic nucleicacid. acid.

10 [0051] 10 [00511 "Expression "Expression cassette" cassette" refers refers to to a polynucleotide comprisinga apromoter a polynucleotidecomprising promoterororother other regulatory sequenceoperably regulatory sequence operably to ato linked linked a sequence sequence encoding encoding a protein. a protein.

[0052]

[00521 The The terms terms "promoter" "promoter" and "expression and "expression control sequence" control sequence" are used are used herein to refer to to herein refer to an array an array of ofnucleic acidcontrol nucleicacid controlsequences sequences that that direct direct transcription transcription ofnucleic of a a nucleic acid. acid. As used As used

herein, aa promoter herein, includes promoterincludes necessary necessary nucleic nucleic acidacid sequences sequences nearstart near the start of the site site of 15 transcription, 15 suchsuch transcription, in the as, the as, in casecase of aof a polymerase polymerase II promoter, II type type promoter, a TATA Aelement. a TATA element. A promoteralso promoter alsooptionally includes optionallyincludes distal distal enhancer enhancer or repressor or repressor elements, elements, can be can which which be located located

muchasasseveral as much as severalthousand basebase thousand pairs pairs fromfrom the start the start sitesite of transcription. of transcription. Promoters Promoters include include

constitutive and constitutive induciblepromoters. andinducible promoters. A "constitutive" A "constitutive" promoter promoter is a promoter that is that is a promoter is active active most environmental under most under environmental and and developmental conditions. An developmentalconditions. An"inducible" promoterisis aa "inducible" promoter 20 promoter 20 promoter is isactive thatthat active under environmentalorordevelopmental underenvironmental regulation. The developmentalregulation. term"operably Theterm "operably linked" refers linked" refers to to aa functional functional linkage between linkagebetween a nucleic a nucleic acidacid expression expression control control sequence sequence

(such as (such promoter,ororarray as aa promoter, arrayofoftranscription transcriptionfactor binding factorbinding sites) sites) andand a second a second nucleic nucleic acid acid wherein sequence,wherein sequence, thethe expression expression control control sequence sequence directs directs transcription transcription of the of the nucleic nucleic acid acid correspondingto to corresponding thesecond the second sequence. sequence.

25 [0053] 25 [0053] A "heterologous A "heterologous sequence" sequence" or a "heterologous or a "heterologous nucleic nucleic acid", acid", as as used used herein,isis one herein, one that originates that fromaasource originates from foreign sourceforeign to to theparticular the host particularhost cell, fromthe cell,or,or,ififfrom thesame same source, source, is is modifiedfrom modified from itsits originalform. original form. Thus, Thus, a heterologous a heterologous expression expression cassette cassette in aiscell in a cell an is an expressioncassette expression thatisis not cassettethat notendogenous endogenous to the to the particular hosthost particular cell, cell, forfor example example by being by being

linked to linked to nucleotide nucleotidesequences sequences from from an expression an expression vectorvector ratherrather than chromosomal than chromosomal DNA, DNA, 30 beingbeing 30 linked linked to a to a heterologous heterologous promoter, promoter, beingtolinked being linked to a reporter a reporter gene, gene, etc. etc.

[0054] The The

[0054] terms terms "nucleic "nucleic acid" acid" are used are and "polynucleotide" and "polynucleotide" used interchangeably interchangeably herein to herein to refer to refer to deoxyribonucleotides deoxyribonucleotides or or ribonucleotides ribonucleotides and polymers and polymers thereofthereof in either in either single-single- or or double-stranded form. double-stranded The term form. The term encompasses nucleicacids encompassesnucleic containing known acids containing nucleotide known nucleotide

13

analogsorormodified analogs modified backbone backbone residues residues or linkages, or linkages, which which are synthetic, are synthetic, naturally naturally occurring, occurring,

and non-naturally and non-naturallyoccurring, occurring, which which havehave similar similar binding binding properties properties as the as the reference reference nucleicnucleic

acid, and acid, and which whicharearemetabolized metabolized in ainmanner a manner similar similar toreference to the the reference nucleotides. nucleotides. Examples Examples

of such of analogsinclude, such analogs include,without without limitation, limitation, phosphorothioates, phosphorothioates, phosphoramidates, phosphoramidates, methyl methyl 5 phosphonates, 5 phosphonates, chiral-methylphosphonates, chiral-methyl phosphonates,2-O-methyl 2-0-methyl ribonucleotides,peptide-nucleic ribonucleotides, peptide-nucleic acids acids (PNAs). (PNAs). 2022204080

[0055]Unless

[0055] Unless otherwise otherwise indicated, indicated, a particular a particular nucleic nucleic acid sequence acid sequence also encompasses also encompasses

conservativelymodified conservatively modified variants variants thereof thereof (e.g., (e.g., degenerate degenerate codon codon substitutions) substitutions) and and complementary complementary sequences, sequences, as well as well as theassequence the sequence explicitly explicitly indicated. indicated. Specifically, Specifically,

10 degenerate 10 degenerate codon codon substitutionsmay substitutions may be be achievedbyby achieved generatingsequences generating sequencesininwhich whichthe thethird third position of position of one oneorormore moreselected selected (or(or all)codons all) codons is substituted is substituted with with mixed-base mixed-base and/orand/or

deoxyinosineresidues deoxyinosine residues (Batzer (Batzer et al.,Nucleic et al., Nucleic Acid Acid Res.Res. 19:5081 19:5081 (1991); (1991); OhtsukaOhtsuka et et al., J. al., J. Biol. Chem. Biol. Chem.260:2605-2608 260:2605-2608 (1985); (1985); Rossolini Rossolini et al.,etMol. al., Cell. Mol. Cell. ProbesProbes 8:91-988:91-98 (1994)). (1994)).

[0056] "Inhibitors,"

[0056] "Inhibitors,""activators," "activators,"andand "modulators" "modulators" of expression of expression or of activity or of activity are toused are used to 15 15 refer refer to inhibitory, to inhibitory, activating, activating, or modulating or modulating molecules, molecules, respectively, respectively, identified identified using using in vitroin vitro and inin vivo and vivo assays assaysfor forexpression expressionor or activityofof activity a a described described target target protein protein (or(or encoding encoding

polynucleotide),e.g., polynucleotide), e.g., ligands, ligands, agonists, agonists,antagonists, antagonists,and andtheir theirhomologs homologs and mimetics. and mimetics. The The term "modulator" term "modulator" includes includes inhibitors inhibitors and and activators. activators. Inhibitors Inhibitors are agents are agents that, that, e.g.,e.g., inhibit inhibit

expressionororbind expression bindto, to,partially partiallyorortotally totally block blockstimulation stimulationororprotease protease inhibitor inhibitor activity, activity,

20 reduce, 20 reduce, decrease, decrease, prevent, prevent, delay delay activation, activation, inactivate, inactivate, desensitize, desensitize, or downorregulate down regulate the the activity of activity of the the described target protein, described target protein, e.g., e.g., antagonists. antagonists. Activators Activatorsareareagents agents that,e.g., that, e.g., induceororactivate induce activate the theexpression expressionof of a described a described target target protein protein or bind or bind to, to, stimulate, stimulate, increase, increase,

open, activate, open, activate, facilitate, facilitate, enhance activationororprotease enhance activation proteaseinhibitor inhibitoractivity, activity,sensitize sensitizeororupup regulate the regulate the activity activity of of described describedtarget targetprotein protein(or (orencoding encoding polynucleotide), polynucleotide), e.g., e.g., agonists. agonists.

25 Modulators 25 Modulators include include naturally naturally occurring occurring and synthetic and synthetic ligands, antagonists ligands, antagonists and(e.g., and agonists agonists (e.g., small chemical small chemicalmolecules, molecules, antibodies antibodies and like and the the like that that function function as either as either agonists agonists or or antagonists). Such antagonists). Suchassays assays forfor inhibitors inhibitors andand activators activators include, include, e.g., e.g., applying applying putative putative

modulatorcompounds modulator compounds to cells to cells expressing expressing the described the described target target proteinprotein and and then then determining determining

the functional the functional effects effects on onthe thedescribed describedtarget targetprotein protein activity,asasdescribed activity, described above. above. Samples Samples or or 30 assays 30 assays comprising comprising described described target protein target protein that arethat are treated treated with a potential with a potential activator, activator, inhibitor, inhibitor,

or modulatorarearecompared or modulator compared to control to control samples samples without without the inhibitor, the inhibitor, activator, activator, or modulator or modulator

to examine to theextent examine the extentof of effect.Control effect. Control samples samples (untreated (untreated with modulators) with modulators) are assigned are assigned a a relative activity relative activity value of 100%. value of 100%.Inhibition Inhibition of of a described a described target target protein protein is achieved is achieved when when the the activity activity value relative to value relative to the the control is about control is 80%,optionally about 80%, optionally 50%50% or 25, or 25, 10%, 10%, 5% or 5% 1%. or 1%.

14

Activation ofthe Activation of thedescribed describedtarget targetprotein proteinis isachieved achieved when when the activity the activity value value relative relative to the to the

control control isis110%, 110%, optionally optionally150%, 150%, optionally optionally200, 300%, 200, 300%,400%, 400%, 500%, 500%, or or 1000-3000% or 1000-3000% or

more higher. more higher.

[0057] Where

[0057] Where chemical chemical substituent substituent groups groups are specified by their by are specified their conventional conventional chemical chemical 5 formulae, 5 formulae, written written from from leftright, left to to right, they they equally equally encompass encompass the chemically the chemically identical identical

substituents that substituents that would wouldresult resultfrom fromwriting writing thethe structure structure from from right right to left,e.g., to left, e.g.,-CHO- -CH is0-2 is 2022204080

equivalent to equivalent to-OCH 2 -. -OCH-.

[0058] TheThe

[0058] termterm "alkyl," "alkyl," by itself by itself or asorpart as part of another of another substituent, substituent, means, means, unlessunless otherwise otherwise

stated, aa straight stated, straight (i.e., (i.e.,unbranched) or branched unbranched) or branchedchain, chain,ororcombination combination thereof, thereof, which which may may be be 10 fully 10 fully saturated, saturated, mono- mono- or polyunsaturated or polyunsaturated and can and can di- include include di- and multivalent and multivalent radicals, radicals, having having the the number of carbon number of atoms designated carbon atoms designated (i.e., (i.e., Ci-Cio meansone C-C means onetoto ten ten carbons). carbons). Examples Examplesofof saturated hydrocarbon saturated hydrocarbon radicals radicals include, include, but but are are not not limited limited to, to, groups groups such such as methyl, as methyl, ethyl,ethyl, n- n propyl, isopropyl,n-butyl, propyl, isopropyl, n-butyl,t-butyl, t-butyl, isobutyl, isobutyl, sec-butyl, sec-butyl, cyclohexyl, cyclohexyl,(cyclohexyl)methyl, (cyclohexyl)methyl, cyclopropylmethyl, cyclopropylmethyl, homologs homologs and isomers and isomers of, forof, for example, example, n-pentyl, n-pentyl, n-hexyl, n-hexyl, n-heptyl,n-heptyl, n- n 15 octyl, 15 octyl, and and the the like. like. An unsaturated An unsaturated alkyl is alkyl group group is one one one having having one double or more or more double bonds or bonds or triple bonds. triple Examples bonds. Examples of unsaturated of unsaturated alkylalkyl groups groups include, include, butnotarelimited but are not limited to, vinyl, to, vinyl, 2- 2 propenyl, crotyl, propenyl, crotyl, 2-isopentenyl, 2-isopentenyl,2-(butadienyl), 2-(butadienyl),2,4-pentadienyl, 2,4-pentadienyl, 3-(1,4-pentadienyl), 3-(1,4-pentadienyl), ethynyl, ethynyl, I- and 1- 3-propynyl,3-butynyl, and 3-propynyl, 3-butynyl, andand the the higher higher homologs homologs and isomers. and isomers.

[0059] TheThe

[0059] termterm "alkylene" "alkylene" by itself by itself or as or as of part partanother of another substituent substituent means means a a divalent divalent

20 radical 20 radicalderived derivedfrom fromananalkyl, alkyl, as as exemplified, exemplified, but but not notlimited, byby limited, -CH 2CH2CH2CH2-. -CHCHCHCH-.

Typically, ananalkyl Typically, alkyl(or (oralkylene) alkylene)group group will will have have fromfrom 1 to I24tocarbon 24 carbon atoms,atoms, with with those those groupshaving groups having10 10 or or fewer fewer carbon carbon atomsatoms being being exemplified exemplified in the present in the present invention. invention. A A "lower alkyl" "lower alkyl"oror"lower "loweralkylene" alkylene" is is a shorter a shorter chain chain alkyl alkyl or alkylene or alkylene group, group, generally generally having having

eight or eight or fewer fewercarbon carbonatoms. atoms.

25 [0060] 25 [0060] The "heteroalkyl," The term term "heteroalkyl," by itself by itself or or inincombination combinationwith withanother anotherterm, term, means, means, unless unless otherwisestated, otherwise stated, aastable stable straight straight or or branched branchedchain, chain,or or cyclic cyclic hydrocarbon hydrocarbon radical, radical, or or combinations combinations thereof,consisting thereof, consisting of at of at least least oneone carbon carbon atoms atoms and and at at least least one heteroatom one heteroatom

selected from selected fromthe thegroup group consisting consisting of N, of O, 0, P, N, SiP, and Si and S, and S, and wherein wherein the nitrogen the nitrogen and and sulfur sulfur atomsmay atoms may optionally optionally be oxidized be oxidized andnitrogen and the the nitrogen heteroatom heteroatom may optionally may optionally be be 30 quaternized. 30 quaternized. The heteroatom(s) The heteroatom(s) 0, N, O, N, P and P and S and S and Si may Si mayatbeanyplaced be placed at any interior interior position position of the heteroalkyl of the groupororatatthe heteroalkyl group theposition positionatatwhich whichthethe alkyl alkyl group group is attached is attached to the to the

remainder of the remainder of the molecule. molecule. Examples include, but Examples include, but are are not notlimited limited to,to, -CH-CH-CH-O-CH, 2 -CH 2 -0-CH 3 ,

CH 2-CH 2-NH-CH 3-CH-CH-N(CH)-CH, CH-CH-NH-CH, , -CH 2-CH 2-N(CH 3)-CH 3-CH-S-CH-CH, , -CH 2-S-CH 2-CH -CH-CH,-S(O)-CH, 3, -CH 2-CH 2 ,-S(O)-CH 3, -CH- -CH 2 CH CH-S(O)-CH, , -CH=CH-0-CH-Si(CH), 2 -S(O) 2 -CH 3-CH=CH-O-CH, 3 , -Si(CH 3 ) 3-CH-CH=N-OCH, , -CH 2-CH=N-OCH 3 , -CH=CH-N(CH 3 )-CH 3 -CH=CH-N(CH)-CH, ,

15

O-CH 3-O-CH2-CH, O-CH, , -O-CH 2-CH 3 , and and -CN.-CN. Up to Up twotoheteroatoms two heteroatoms may bemay be consecutive, consecutive, such such as, as, for for example, -CH-NH-OCH example, -CH 2-NH-OCH 3 and -CH 2 -0-Si(CH and -CH-O-Si(CH). 3) 3 . Similarly, Similarly, the term the term "heteroalkylene" "heteroalkylene" by by itself ororasaspart itself partof ofanother another substituent substituent means means a adivalent divalentradical radicalderived derived from from heteroalkyl, heteroalkyl, as as exemplified, butbut exemplified, not not limited by, -CH limited 2-CH by, 2-S-CH 2-CH 2- and -CH-CH-S-CH-CH- -CH and 2 -S-CH 2-CH 2-NH-CH 2-. -CH-S-CH-CH-NH-CH.

55 For For heteroalkylene heteroalkylene groups, groups, heteroatoms heteroatoms can alsocan alsoeither occupy occupy or either both ofor both the of the chain chain termini termini (e.g., alkyleneoxy, (e.g., alkylenedioxy,alkyleneamino, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, alkylenediamino, and theand the Still like). like). Still 2022204080

further, for further, for alkylene and heteroalkylene alkylene and heteroalkylene linking linking groups, groups, no orientation no orientation of linking of the the linking groupgroup is is impliedbybythe implied thedirection directionininwhich whichthethe formula formula of the of the linking linking group group is written. is written. For example, For example,

the formula the formula -C(O) 2 R'-represents -C(O)R'- represents both -C(O) 2 R'-and -C(O)R'- and-R'C(O)-. -RC(O) 2 -. As As described described above, above,

10 heteroalkyl 10 heteroalkyl groups, groups, as herein, as used used herein, include include those groups those groups that arethat are attached attached to the remainder to the remainder of of the molecule the molecule through a heteroatom, through such such a heteroatom, as -C(O)R', -C(O)NR', as -C(O)R', -NR'R", -NR'R, -C(O)NR', -OR', -SR, and/or -OR', - -SR, and/or SO R'.Where SOR'. 2 Where "heteroalkyl" "heteroalkyl" is recited, is recited, followed followed by recitations by recitations of specific of specific heteroalkyl heteroalkyl groups, groups,

such such as as -NR'R" -NR'Rororthe thelike, like,itit will will be be understood understoodthat thatthetheterms heteroalkyl terms and and heteroalkyl -NR'R" are not -NR'R" are not redundantorormutually redundant mutually exclusive. exclusive. Rather, Rather, the specific the specific heteroalkyl heteroalkyl groupsgroups are recited are recited to add to add 15 clarity. 15 clarity. Thus, Thus, the term the term "heteroalkyl" "heteroalkyl" shouldshould not be not be interpreted interpreted herein herein as as excluding excluding specific specific heteroalkyl heteroalkyl groups, groups,such suchas as -NR'R" -NR'Ror the like. or the like.

[00611TheThe

[0061] terms terms "cycloalkyl" "cycloalkyl" and "heterocycloalkyl", and "heterocycloalkyl", by themselves by themselves or in combination or in combination

with other with otherterms, terms,represent, represent,unless unlessotherwise otherwise stated, stated, cyclic cyclic versions versions of "alkyl" of "alkyl" and and "heteroalkyl", respectively. "heteroalkyl", respectively. Additionally, Additionally, forfor heterocycloalkyl, heterocycloalkyl, a heteroatom a heteroatom can occupy can occupy the the 20 position 20 position at which at which the heterocycle the heterocycle is attached is attached to the to the remainder remainder of the molecule. of the molecule. Examples Examples of of cycloalkylinclude, cycloalkyl include,but butare arenot notlimited limitedto,to,cyclopentyl, cyclopentyl,cyclohexyl, cyclohexyl, I-cyclohexenyl, 1-cyclohexenyl, 3- 3 cyclohexenyl,cycloheptyl, cyclohexenyl, cycloheptyl, andand the the like. like. Examples Examples of heterocycloalkyl of heterocycloalkyl include,include, but are but not are not limited to, limited to, I1 -(1,2,5,6-tetrahydropyridyl), 1-piperidinyl,2-piperidinyl, -(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 3-piperidinyl, 4- 4 morpholinyl,3-morpholinyl, morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-2-yl,

25 tetrahydrothien-3-yl, 25 tetrahydrothien-3-yl, I -piperazinyl, 1 -piperazinyl, 2-piperazinyl, 2-piperazinyl, and theand theA like. like. A "cycloalkylene" "cycloalkylene" and and "heterocycloalkylene"refer "heterocycloalkylene" refer to to a divalent a divalent radical radical derived derived fromfrom cycloalkyl cycloalkyl and and heterocycloalkyl,respectively. heterocycloalkyl, respectively.

[0062] TheThe

[0062] terms terms "halo" "halo" or "halogen," or "halogen," by themselves by themselves or of or as part as another part of substituent, another substituent, mean,unless mean, unlessotherwise otherwise stated, stated, a fluorine, a fluorine, chlorine, chlorine, bromine, bromine, or iodine or iodine atom.atom. Additionally, Additionally,

30 terms 30 terms such such as as "haloalkyl,"are "haloalkyl," aremeant meanttoto include include monohaloalkyl monohaloalkyland andpolyhaloalkyl. polyhajoalkyl. For For example,the example, theterm term"halo(C-C)alkyl" "halo(C-C )alkyl" is meanis to 4 mean to include, include, but not but not be limited be limited to, to, trifluoromethyl, 2,2,2-trifluoroethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 4-chlorobutyl,3-bromopropyl, 3-bromopropyl, andlike. and the the like.

[0063] TheThe

[0063] termterm "aryl" "aryl" means, means, unlessunless otherwise otherwise stated, stated, a polyunsaturated, a polyunsaturated, aromatic,aromatic,

hydrocarbon hydrocarbon substituent substituent which which cana be can be a single single ring ring or multiple or multiple rings rings (preferably (preferably from 1 from to 3 I to 3 16

rings) which rings) arefused whichare fusedtogether or or together linked linked covalently. The The covalently. term term "heteroaryl" refers refers "heteroaryl" to to aryl aryl (orrings) groups(or groups rings) that that contain containfrom to to fromoneone four four heteroatoms heteroatoms selected selected from from and 0, N, O, N, S, and S, wherein nitrogenandand thenitrogen wherein the sulfur sulfur atoms are are atoms optionally optionally oxidized, oxidized, andnitrogen and the the nitrogen atom(s) atom(s) are are optionally quaternized. optionally quaternized.A heteroaryl A heteroaryl group group canattached can be be attached the remainder to thetoremainder of the molecule of the molecule

5 through 5 through a carbon a carbon or or heteroatom.Non-limiting heteroatom. Non-limiting examples examples of of aryland aryl andheteroaryl groups heteroaryl groups include phenyl, include phenyl,1-naphthyl, 2-naphthyl, I-naphthyl,2-naphthyl, 4-biphenyl, 4-biphenyl, 1-pyrrolyl, 1-pyrrolyl, 2-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrrolyl, 3- 3 2022204080

pyrazolyl, 2-imidazolyl, pyrazolyl, 4-imidazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, pyrazinyl, 2-oxazolyl, 2-oxazolyl, 4-oxazolyl, 4-oxazolyl, 2-phenyl-4 2-phenyl-4-

3-isoxazolyl,4-isoxazolyl, 5-oxazolyl,3-isoxazolyl, oxazolyl, 5-oxazolyl, oxazolyl, 4-isoxazolyl, 5-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 2-thiazolyl, 4-thiazolyl, 4-thiazolyl, 5- 5 thiazolyl, 2-furyl, thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyridyl, 3-pyridyl, 2-pyrimidyl,4- 4 4-pyridyl,2-pyrimidyl, 10 pyrimidyl, 10 pyrimidyl, 5-benzothiazolyl, 5-benzothiazolyl, purinyl, purinyl, 2-benzimidazolyl, 2-benzimidazolyl, 5-indolyl, 5-indolyl, 1-isoquinolyl, 1-isoquinolyl, 5- 5 2-quinoxalinyl,5-quinoxalinyl, isoquinolyl, 2-quinoxalinyl, isoquinolyl, 5-quinoxalinyl, and and 3-quinolyl, 3-quinolyl, 6-quinolyl. 6-quinolyl. Substituents Substituents for for each each of the of abovenoted the above notedaryl aryland andheteroaryl heteroaryl ring ring systems systems are selected are selected from from the group the group of acceptable of acceptable

substituents described substituents below. describedbelow. "Arylene" "Arylene" and "heteroarylene" and "heteroarylene" refers refers to to a divalent a divalent radicalradical

derived from derived froma aaryl aryland andheteroaryl, heteroaryl,respectively. respectively.

15 15 [0064]

[0064] For brevity, For brevity, the the term term "aryl" "aryl" when when used used in in combination combination with with otherterms other (e.g., terms(e.g., aryloxy, arylthioxy, aryloxy, includesboth arylalkyl)includes arylthioxy,arylalkyl) both aryl andand aryl heteroaryl heteroaryl rings rings as defined as defined above. above.

Thus,the Thus, theterm "arylalkyl"isismeant term"arylalkyl" meant to to include include those those radicals radicals in which in which an aryl an aryl groupgroup is is attached toto ananalkyl attached alkyl group (e.g.,benzyl, group(e.g., benzyl,phenethyl, phenethyl, pyridylmethyl pyridylmethyl andlike) and the the like) including including

those alkyl those groupsininwhich alkyl groups which atomatom a carbon a carbon (e.g., (e.g., a methylene group)group) a methylene has been been replaced hasreplaced by, for by, for example, 20 example, 20 an oxygen an oxygen atomatom (e.g., (e.g., phenoxymethyl, phenoxymethyl, 2-pyridyloxymethyl, 2-pyridyloxymethyl, 3-(1 3-(1-

naphthyloxy)propyl, naphthyloxy)propyl, andand the the like). like).

[0065] TheThe

[0065] term"oxo" term "oxo" as as hereinmeans usedherein used meansananoxygen oxygen thatisis double that bondedtoto aa carbon double bonded carbon atom. atom.

The

[0066] The

[0066] "alkylsulfonyl"asas used term"alkylsulfonyl" term used herein meansaa moiety herein means having the moiety having the formula -S(02) formula -S(O2)-

25 R',R', R' R'is where where is ananalkyl alkyl group as defined group as defined above. R'may above. R' may have specified number haveaa specified of carbons number of carbons

(e.g. (e.g. "C1-C4 alkylsulfonyl"). "C1-C4 alkylsulfonyl").

[0067] EachEach

[0067] of above of the the above (e.g., (e.g., terms terms "alkyl," "alkyl," "heteroalkyl," "heteroalkyl," "aryl""aryl" and "heteroaryl") and "heteroaryl") are are meanttotoinclude meant includeboth both andand substituted substituted unsubstituted unsubstituted formsforms of theof the indicated indicated radical. radical.

Exemplary Exemplary for for substituents substituents each each typetype of radical of radical are are provided provided below.below.

30 [0068] 30 [00681 Substituents Substituents for theand for the alkyl alkyl and heteroalkyl heteroalkyl radicals (including radicals (including those those groups groups often often referred to referred to as as alkylene, alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, alkynyl, cycloalkyl, cycloalkyl,

heterocycloalkyl, cycloalkenyl, heterocycloalkyl,cycloalkenyl, andand can becan heterocycloalkenyl) heterocycloalkenyl) onebe or one moreor ofmore of a variety a variety of of selectedfrom, groupsselected groups notnot from,butbut limited limited to:to: -OR', =0,=0, -OR', =NR', =NR', =N-OR', -NR'R",-NR'R", =N-OR', -SR', -halogen, -SR', -halogen, -

17

SiR'R"R"', -OC(O)R', SiR'R"R", -OC(O)R',-C(O)R', -C(O)R',-COR', -CO 2R', -CONR'R", -CONR'R", -OC(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR"C(O)R',

-NR'-C(O)NR"R"', -NR"C(O)R', -NR'-C(O)NR"R", -NR"C(O) 2 R',-NR-C(NR'R"R"")=NR"" -NR-C(NR'R"R.')=NR"", -NR-C(NR'R")=NR', -NR-C(NR'R")=NR", -S(O)R', -S(O)R',-

S(O) 2R', -S(O)NR'R", S(O)R', -S(O)2NR'R",-NRSOR', -NRSO-CN 2R', and -CN-NO andin -NO 2 in a number a number rangingranging fromtozero from zero to (2m'+1), (2m'+1),

wherem'm'is where thetotal is the total number number of of carbon carbon atoms atoms in such in such radical. radical. R',R"R",andR"R""and R', R", R"" each each 5 preferably 5 preferably independently independently refer refer to to hydrogen, hydrogen, substituted substituted or unsubstituted or unsubstituted heteroalkyl, heteroalkyl,

substituted or substituted or unsubstituted unsubstitutedcycloalkyl, cycloalkyl,substituted substituted or or unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, 2022204080

or unsubstituted substituted or substituted unsubstitutedaryl (e.g., aryl aryl(e.g., substituted with aryl substituted with1-3 1-3halogens), halogens),substituted substituted or or unsubstitutedalkyl, unsubstituted alkoxyororthioalkoxy alkyl,alkoxy thioalkoxy groups, groups, or arylalkyl or arylalkyl groups. groups. When aWhen a compound compound of of the invention the invention includes includesmore more than than one one R group, R group, for example, for example, each ofeach the of the R is R groups groups is 10 independently 10 independently selectedasasare selected areeach eachR', R', R", R", R"' and R"" R" and R"" groups groups when whenmore morethan thanone oneofofthese these groupsisis present. groups present. When When R'and R' and R" attached R" are to thetosame are attached the nitrogen same nitrogen atom, atom, they canthey be can be combined combined with with thethe nitrogen nitrogen atomatom to form to form a 4-, a5-, 4-,6-, 5-, or 6-,7-membered or 7-membered ring. ring. For For example, example,

-NR'R"isismeant -NR'R" meantto to include, include, butbut notnot be be limited limited to, to,1-pyrrolidinyl 1-pyrrolidinyl and and 4-morpholinyl. 4-morpholinyl. From From the above the abovediscussion discussionof of substituents, substituents, oneone of of skillin inthetheartartwill skill willunderstand understand that that thethe term term

15 "alkyl" 15 "alkyl" is meant is meant to include to include groups groups including including carbonbound carbon atoms atoms bound other to groups to groups than other than hydrogen groups, hydrogen groups, such such as haloalkyl (e.g., as haloalkyl (e.g.,-CF-CF 3 and and-CH 2CF 3and -CHCF) ) and (e.g., -C(O)CH acyl(e.g., acyl -C(O)CH, 3

, -C(O)CF 3, -C(O)CHOCH, -C(O)CF, -C(O)CH 2 OCH 3, and and thethe like). like).

[0069] Similar

[0069] Similar to the to the substituents substituents described described foralkyl for the the alkyl radical, radical, substituents substituents foraryl for the the aryl and heteroaryl and heteroarylgroups groupsareare varied varied andand are are selected selected from, from, for example: for example: halogen, halogen, -OR', -NR'R", -OR', -NR'R",

20 -SR', 20 -SR', -halogen,-SiR'R"R", -halogen, -SiR'R"R"',-OC(O)R', -OC(O)R', -C(O)R', -C(O)R', -CO 2-CONR'R", -COR', R', -CONR'R", -OC(O)NR'R", -OC(O)NR'R",

-NR"C(O)R', -NR'-C(O)NR"R', -NR"C(O)R', -NR"C(O) 2 R',-NR-C(NR'R"R")=NR"", -NR'-C(O)NR"R", -NR"C(O)R', -NR-C(NR'R"R')=NR"" -NR-C(NR'R")=NR"', -NR-C(NR'R")=NR", -S(O)R', -S(O)R', -S(O) 2-S(O)NR'R", -S(O)R', R', -S(O) 2NR'R", -NRSO -NRSOR', R', -CN -CN 2and -NO, and -NO -R', -N,2 , -R', -N 3 , -CH(Ph), , fluoro(CI-C 4)alkoxy, -CH(Ph) 2fluoro(C-C)alkoxy, andand fluoro(C-C 4)alkyl, fluoro(C-C)alkyl, a number in ainnumber ranging ranging zerozero fromfrom to the to the

total number total number ofof open open valences valences on the on the aromatic aromatic ring system; ring system; and R', and where where R', and R", R" R", R"" R"'are and R"" are 25 preferably 25 preferably independently independently selected selected from hydrogen, from hydrogen, substituted substituted or unsubstituted or unsubstituted alkyl, alkyl, substituted or substituted or unsubstituted unsubstitutedheteroalkyl, heteroalkyl,substituted substituted or or unsubstituted unsubstituted cycloalkyl, cycloalkyl, substituted substituted or or unsubstituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted arylaryl and and substituted substituted or or unsubstitutedheteroaryl. unsubstituted heteroaryl.When When a compound a compound of the invention of the invention includes includes more thanmore one R than one R group, for group, forexample, example,each each of of thethe R groups R groups is independently is independently selected selected as are as areR', each each R",R', R" R", and R'" and 30 R""R"" 30 groups groups whenwhen moremore than than onethese one of of these groups groups is present. is present.

[0070] Two Two

[0070] ofsubstituents of the the substituents on adjacent on adjacent atoms atoms of of the the aryl or aryl or heteroaryl heteroaryl ring mayring may optionally form optionally forma aring ringofofthe theformula formula -T-C(O)-(CRR')q-U-, -T-C(O)-(CRR')q-U-, whereinwherein T and U T areand U are independently -NR-, independently -NR-, -0-,-0-, -CRR'- -CRR'- or a or a single single bond,bond, and q and is anq integer is an integer of fromof0 from to 3. 0 to 3.

Alternatively, two Alternatively, twoofofthe substituentsonon thesubstituents adjacent adjacent atoms atoms of aryl of the the aryl or heteroaryl or heteroaryl ring ring may may

18

optionally be replaced optionally be replacedwith a substituent witha substituent thethe of of formula formula -A-(CH)-B-, )r-B-, wherein -A-(CH 2wherein A and A and B are B are -CRR'-, independently-CRR'-, independently -0-, -NR-, -0-, -NR-, -S-, -S-, -S(O)-, -S(O)-, -S(O) -S(O)-, 2 -, -S(O) 2 NR'- or -S(O)NR'- or a single bond,a single andbond, r is and r is integer of an integer an from1 Itoto4.4.One offrom Oneof of thethe single single bonds bonds of the of the ring ring new new so formed so formed may optionally may optionally

be replaced be replacedwith double witha adouble bond. bond. Alternatively, Alternatively, two of the substituents twotheofsubstituents on adjacent on adjacent atoms atoms of of arylaryl 55 the the ringring or heteroaryl or heteroaryl may may optionally optionally be replaced be replaced with a with a substituent substituent of the formula of the formula

-(CRR')s-X'-(C"R"')d-, -(CRR')-X'-(C"R")d-, where where S ands dand are dindependently are independently integers integers of from of from 0 to 0 toX'3, is 3, and and-0-, X' is -0-, 2022204080

-NR'-, -S-, -NR'-, -S(0)2-,oror-S(O)NR'-. -S(O)-, -S(O)-, -S-, -S(O)-, -S(O) 2 NR'-. R, R', R, The substituents The substituents R" R', and R" R" are R"' and preferably are preferably selected independentlyselected independently from from hydrogen, hydrogen, substituted substituted or unsubstituted alkyl, alkyl, or unsubstituted substituted substituted or or substitutedor orunsubstituted cycloalkyl,substituted unsubstitutedcycloalkyl, unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or or 10 unsubstituted 10 unsubstituted aryl,aryl, and substituted and substituted or unsubstituted or unsubstituted heteroaryl. heteroaryl.

[00711 As As

[0071] usedused herein, herein, the term the term "heteroatom" "heteroatom" orheteroatom" or "ring "ring heteroatom" is meant is meant to includeto include oxygen(O), oxygen (0),nitrogen nitrogen (N), (N), sulfur sulfur (S),phosphorus (S), (P), (P), phosphorus and and silicon silicon (Si).(Si).

[0072] A "substituent

[0072] A "substituent group," group," as used as used herein, herein, means means group selected a group aselected from the from the following following

moieties: moieties:

15 15 (A) -OH, -OH, (A) -SH,2,-CN, -NH, -NH -SH, -CF, -CN,-NO, , -NOhalogen, -CF3oxo, 2 , oxo, halogen, unsubstituted unsubstituted alkyl, alkyl, unsubstituted unsubstituted

heteroalkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, cycloalkyl,unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl,

unsubstituted aryl, unsubstituted unsubstituted aryl, unsubstitutedheteroaryl, heteroaryl,andand

(B) (B) alkyl, heteroalkyl, alkyl, cycloalkyl, heterocycloalkyl, heteroalkyl, cycloalkyl, aryl,andand heterocycloalkyl,aryl, heteroaryl, heteroaryl, substituted withwith substituted at least at one substituent least one selected from: substituent selected from:

20 20 (i) (i) oxo, -OH, -NH, -NH 2-SH, , -SH,-CN, -CN,-CF, -CF-NO, 3, -NOhalogen, 2, halogen, unsubstituted unsubstituted alkyl, alkyl,

unsubstitutedheteroalkyl, unsubstituted unsubstituted heteroalkyl,unsubstituted cycloalkyl, cycloalkyl, unsubstituted unsubstituted

unsubstituted heterocycloalkyl,unsubstituted heterocycloalkyl, aryl, unsubstituted aryl,unsubstituted heteroaryl, heteroaryl, and and

(ii) (ii) alkyl, alkyl, heteroalkyl, heteroalkyl, cycloalkyl, cycloalkyl, heterocycloalkyl, aryl, aryl, heterocycloalkyl, and heteroaryl, and heteroaryl,

withatat least substituted with substituted least one substituentselected one substituent from: selectedfrom:

25 25 (a) oxo,oxo, (a) -OH, -OH, -NH,-NH 2, -SH, -SH, -CN, -CN, -NO,3, halogen, -CF, -CF -NO 2, halogen, unsubstituted unsubstituted

alkyl, unsubstituted alkyl, heteroalkyl,unsubstituted unsubstituted heteroalkyl, unsubstituted cycloalkyl, cycloalkyl,

heterocycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted unsubstituted unsubstituted aryl, aryl,

unsubstitutedheteroaryl, unsubstituted heteroaryl,and and

alkyl, (b) alkyl, (b) heteroalkyl, heteroalkyl, cycloalkyl, cycloalkyl, heterocycloalkyl, heterocycloalkyl, aryl, aryl, or or 30 30 heteroaryl, substituted heteroaryl, withatatleast substitutedwith least one substituentselected onesubstituent selected from oxo, from oxo, -OH, -NH 2-SH, -OH, -NH, , -SH,-CN, -CN,-CF, 3, -NO -CF-NO, 2, halogen, halogen,

unsubstitutedalkyl, unsubstituted alkyl,unsubstituted unsubstitutedheteroalkyl, heteroalkyl, unsubstituted unsubstituted

19

cycloalkyl, unsubstituted cycloalkyl, heterocycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted unsubstituted aryl, aryl,

and unsubstituted and unsubstitutedheteroaryl. heteroaryl.

[0073] A "size-limited substituent"

[0073] A "size-limited substituent" or " size-limited or size-limited substituent group," group," substituent as used as used herein herein meansa agroup means group selected selected from from all all of the of the substituents substituents described aboveabove described for a for a "substituent "substituent group," group,"

5 wherein 5 wherein each each substituted substituted or unsubstituted or unsubstituted alkyl alkyl is is a substituted a substituted or unsubstituted or unsubstituted C-C alkyl, C-C 20 alkyl,

2022204080 each substitutedororunsubstituted each substituted is is heteroalkyl unsubstitutedheteroalkyl a substituted or or a substituted unsubstituted unsubstituted to 2 to2 20 20 membered membered heteroalkyl, eacheach heteroalkyl, substituted substituted or unsubstituted or unsubstituted cycloalkyl cycloalkyl is a substituted is a substituted or or unsubstitutedC-C unsubstituted -C 8 cycloalkyl, C 4 cycloalkyl, and each each substituted and substituted or unsubstituted or unsubstituted heterocycloalkyl heterocycloalkyl is a is a substituted unsubstituted4 4toto8 8membered or unsubstituted substituted or membered heterocycloalkyl. heterocycloalkyl.

[00741 10 [0074] 10 A "lower A "lower substituent" substituent" or "lower or "lower substituent substituent group," as as group," usedherein used meansa agroup hereinmeans group selected from selected all of fromall ofthe the substituents above describedabove substituentsdescribed for for a "substituent a "substituent group," group," wherein wherein each each substituted unsubstitutedalkyl or unsubstituted substituted or substitutedororunsubstituted alkylisisa asubstituted unsubstitutedC-CC-C 8 alkyl, alkyl, each each or unsubstituted substituted or substituted unsubstitutedheteroalkyl substitutedororunsubstituted heteroalkylis isa asubstituted unsubstituted 2 8tomembered 2 to 8 membered heteroalkyl, each heteroalkyl, eachsubstituted substitutedororunsubstituted unsubstituted cycloalkyl cycloalkyl is aissubstituted a substituted or unsubstituted or unsubstituted C- C5

C7 cycloalkyl, 15 C cycloalkyl, 15 and each each substituted andsubstituted or unsubstituted or unsubstituted heterocycloalkyl heterocycloalkyl is a substituted is a substituted or or unsubstituted5 5toto77membered unsubstituted membered heterocycloalkyl. heterocycloalkyl.

[0075] termterm

[00751 The The "pharmaceutically "pharmaceutically salts" issalts" acceptable acceptable meant meantisto include include to salts of salts of the the active active compounds compounds which which are prepared are prepared with relatively with relatively nontoxic acids oracids nontoxic bases, bases, depending or depending on the on the particularsubstituents particular substituentsfound thethe on on found describedherein. compoundsdescribed compounds herein. When compounds When compounds of the of the 20 present 20 present invention invention contain contain relatively acidicacidic relatively functionalities, functionalities, base addition base addition salts salts can be can be obtained obtained

by by contacting neutralform theneutral contactingthe of of form such such compounds with a with compounds sufficient amount amount a sufficient of the desired of the desired

base, either neat base, either or in neat or suitable inert in aa suitable inert solvent. Examples solvent. Examples of of pharmaceutically pharmaceutically acceptable base base acceptable addition saltsinclude addition salts sodium, include potassium, sodium, calcium, potassium, ammonium, calcium, amino, or organic amino, ammonium, organic ormagnesium magnesium

salt, or salt, oraasimilar salt.When similar salt. compounds When compounds of present of the the present invention invention contain contain relatively relatively basic basic 25 functionalities, 25 acidacid functionalities, addition addition can can saltssalts be obtained be obtained by contacting by contacting the neutral form of form the neutral such of such compounds compounds withwith a sufficient a sufficient amount of theof amount the desired desired eithereither acid, acid, neat neat or in or a suitable inert inert in a suitable Examples solvent. Examples solvent. of pharmaceutically of pharmaceutically acceptable acceptable acid addition acid addition salts include salts include those those derived derived frominorganic from inorganicacids acidslike likehydrochloric, hydrochloric, hydrobromic, hydrobromic, nitric, nitric, carbonic, carbonic,

monohydrogencarbonic,phosphoric, monohydrogencarbonic, phosphoric,monohydrogenphosphoric monohydrogenphosphoric, dihydrogenphosphoric, dihydrogenphosphoric,

30 sulfuric, 30 sulfuric, monohydrogensulfuric, monohydrogensulfuric, hydriodic, hydriodic, or phosphorous or phosphorous acids acids and the andasthe like, well as as like, thewell as the salts derived salts fromrelatively derived from relativelynontoxic nontoxicorganic organic acids acids likelike acetic, acetic, propionic, propionic, isobutyric, isobutyric, maleic, maleic,

malonic,benzoic, malonic, succinic,suberic, benzoic,succinic, fumaric, suberic,fumaric, lactic, mandelic, lactic,mandelic, phthalic, phthalic, benzenesulfonic, benzenesulfonic, p- p tolylsulfonic, citric, tartaric, tolylsulfonic, citric, tartaric,methanesulfonic, thelike. andthe methanesulfonic, and Alsoincluded like. Also areare included salts salts of of amino amino

such asasarginate acids such acids like,and andthethelike, arginateand andsalts organicacids saltsofoforganic acids likeglucuronic like glucuronic or galactunoric or galactunoric

20

acids and acids andthe the like (see, for like (see, for example, Berge example,Berge et et al.,"Pharmaceutical al., "Pharmaceutical Salts", Salts", Journal of of Journal Pharmaceutical Pharmaceutical Science, Science, 1977, 1977, 66, 1-19). 66, 1-19). Certain Certain specific specific compounds compounds of the of the present present invention bothbasic containboth invention contain basic and and acidic acidic functionalities functionalities that that allow the the allow compounds to be to be compounds convertedinto converted baseororacid eitherbase intoeither acidaddition salts. additionsalts.

5 [0076] 5 Thus,Thus,

[0076] the compounds the present of theofpresent the compounds invention may may invention exist exist as salts with as saltswith

2022204080 acceptable pharmaceuticallyacceptable pharmaceutically acids. acids. The present The present invention invention includes includes such salts. salts. Examples such Examples of of such salts include such salts include hydrochlorides, hydrochlorides,hydrobromides, hydrobromides, sulfates, sulfates, methanesulfonates, methanesulfonates, nitrates, nitrates,

maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates, (-)-tartrates or mixtures maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates, (-)-tartrates or mixtures

thereof racemic includingracemic thereof including mixtures, mixtures, succinates, succinates, benzoates benzoates and salts and salts with amino with amino acidsas such acids such as 10 glutamic 10 glutamic These These acid.acid. saltsbemay salts may be prepared prepared byknown by methods methods known to those to those skilled skilled in the art. in the art.

[00771 The The

[0077] formsforms neutral neutral of the the compounds ofcompounds are preferably are preferably regenerated regenerated by contacting by contacting the the salt with salt a base with a or acid base or andisolating acid and parentcompound theparent isolatingthe compound in conventional in the the conventional manner. manner. The The parent formofofthe parent form thecompound compound differs differs from from the various the various salt forms salt forms in certain in certain physical physical

properties, solubility inin polar suchasas solubility properties, such solvents. polar solvents.

15 [0078] 15 [0078] In addition In addition to saltforms, to salt thepresent forms,the invention provides present invention provides compounds, whichare compounds, which in aa arein form.Prodrugs prodrugform. prodrug Prodrugs of the of the compounds compounds described described herein are thoseare herein compounds that readilythat those compounds readily undergochemical undergo chemical changes changes underunder physiological physiological conditions conditions to provide to provide the compounds the compounds of the of the present invention. present Additionally, invention.Additionally, prodrugs prodrugs canconverted can be to the to be converted the compounds compounds of the of the present present invention bybychemical invention chemicalor or biochemical biochemical methods methods in vivo in an ex vivo environment. an exenvironment. For For example, example, cancan prodrugs 20 prodrugs 20 be be slowly slowly converted converted to to thecompounds the compounds of the of the present inventionwhen presentinvention placedininaa whenplaced patchreservoir transdermalpatch transdermal reservoir with with a suitable a suitable enzyme enzyme or chemical or chemical reagent. reagent.

Certain 10079] Certain

[0079] of the of compounds compounds the present present invention can existcan invention in exist in unsolvated unsolvated forms forms as well as as well as solvated includinghydrated forms,including solvated forms, hydrated forms. forms. In general, In general, the solvated forms forms the solvated are equivalent are equivalent to to forms unsolvatedforms unsolvated andand areare within within encompassed encompassed theofscope the scope of the present the present invention. invention. Certain Certain compounds 25 compounds 25 of the of the present present invention invention maymay exist exist in in multiple crystalline or multiplecrystalline or amorphous forms. In amorphous forms. In general, all physical general, all formsare physical forms equivalentforfor areequivalent thethe uses uses contemplated contemplated by the the present bypresent invention invention

and are and intendedtotobebewithin are intended withinthethescope scope of the of the present present invention. invention.

Certaincompounds

[0080] Certain

[0080] compoundsof of thethe inventionpossess presentinvention present asymmetriccarbon possess asymmetric atoms carbonatoms (optical centers) (optical or double centers) or bonds;thetheracemates, doublebonds; racemates, diastereomers, diastereomers, tautomers, tautomers, geometric isomers isomers geometric 30 andand 30 isomers individualisomers individual areencompassed are encompassed within within thethe scope scope of of thepresent the invention. The present invention. The compounds of the compounds of the present present invention invention doinclude do not those those not include which which are areinknown known the artintothe tooto be too be art

unstable to synthesize unstable to synthesizeand/or and/orisolate. isolate.

21

[00811 TheThe

[0081] compounds compounds of the of the present present invention may inventionmay also containunnatural alsocontain proportions of unnatural proportions of

atomicisotopes atomic isotopesatatone oneorormore more of of thethe atoms atoms thatthat constitute constitute suchsuch compounds. compounds. For example, For example,

the compounds the compounds may may be radiolabeled be radiolabeled with radioactive with radioactive isotopes, such as such isotopes, as for example for example tritium tritium (³H), iodine-125 (¹²I) or carbon-14 (¹C). All isotopic variations of the compounds (3H), iodine-125 (1I) or carbon-14 (1C). All isotopic variations of of the the compounds of the present 55 present invention, invention, whether whether radioactive or not,orare radioactive not, encompassed scope the within thewithin are encompassed scope of the of the

present invention. present invention. 2022204080

BRIEF DESCRIPTION BRIEF OF THE DESCRIPTION OF DRAWINGS THEDRAWINGS

[0082] Figure 1. Compound

[00821 Figure 1. Compound for seven for treatmenttreatment seven days is sufficient to induce to days is sufficient induce pluripotent pluripotent

10 stemstem 10 cells from cellsfrom human human fibroblasts withthe transducedwith fibroblaststransduced thefour reprogrammingfactors. four reprogramming (a) factors. (a) Timeline for human Timeline for iPSCinduction human iPSC using combined induction using SB431542 combinedSB431542 andand PD0325901 PD0325901 treatment treatment

along with4TFs. alongwith beganbegan Treatment 4TFs.Treatment withre-seeding with cell at day at cell re-seeding day 74TF 7 after after 4TF transduction transduction and and was maintained wasmaintained forfor (b) (b) 7 days. 7 days. Staining for for Staining ALP ALP+ colonies colonies that emerged that emerged in the untreated in the untreated

(left) or 22 compound-treated (left) or (right)cultures compound-treated (right) cultures within within seven seven days. days. (c) RT-PCR showing showing (c) RT-PCR 15 elevated 15 elevated endogenous mRNAmRNA endogenous expression expression of pluripotency of pluripotency markers OCT4 OCT4 markers and NANOG and NANOG in 2 in 2 compound-treated compound-treated cultures. cultures. (d) TRA-1-81 (d) TRA-1-81 staining staining at day at day 14 14 without without with or (left) or(left) with 2(right) (right) 2 (e) The treatment. (e) compoundtreatment. compound Thenumbers NANOG* numbersofofNANOG+ colonies colonies at day at day 14 under 14 under different different

conditionsareareplotted. treatmentconditions treatment Typical plotted.(f)(f)Typical staining staining for for hESC-specific hESC-specific markers markers (NANOG (NANOG and SSEA4) and SSEA4) exhibited exhibited by D14 D14 iPSCs. by iPSCs. Scale50bars, Scale bars, 50(dpm µm in in & f). (d & f).

[00831 20 [0083] 20 Figure Figure 2. Prolonged 2. Prolonged compound compound treatment treatment and cell cell passaging andpassaging dramatically dramatically increased increased

the number the of reprogrammed number of colonies. (a) reprogrammedcolonies. of human Timeline of (a) Timeline human iPSC using inductionusing iPSCinduction SB431542, PD0325901 SB431542,PD0325901 and and thiazovivin. 30 30 thiazovivin.(b)(b)DayDay iPSCs iPSCs expressed expressed pluripotency pluripotency markers markers

NANOG, NANOG, SSEA4 SSEA4 Scale Scale and TRA-1-81. and TRA-1-81. pmALP 50 µm50(c) bars, bars, ALP staining (c) staining of day of day 30 cultures 30 cultures with with

(upper without(lower panels)ororwithout (upperpanels) (lower panels) panels) 3 compound 3 compound treatment. treatment. Boxed Boxed areas areas in the in left the left 25 panels 25 areare panels enlargedininthe enlarged theright Scale bars, panels. Scale right panels. bars,200 200pm µm (d) (d)Number of NANOG* Number of colonies NANOG+ colonies

on day 30 under different treatment conditions, on day 30 under different treatment without conditions, splitting. without (e) Number (e) Number splitting. of NANOG* of NANOG+

coloniesononday colonies from day3030from 3 compound-treated 3 compound-treated cultures cultures trypsinized trypsinized as indicated. as indicated. (f) (f) RT-PCR RT-PCR on iPSC on coloniesobtained iPSCcolonies withwith obtained 3 compound 3 compound treatment treatment shows reactivated expressionexpression shows reactivated of of pluripotency markers. endogenouspluripotency endogenous HDF: markers. HDF: Human Human Dermal Dermal Fibroblast. Fibroblast.

30 30 100841

[0084] Figure In vitro 3. In 3.vitro Figure and and in vivo in vivo differentiation ofofiPSCs differentiation with 33 compound generated with iPSCsgenerated compound (a) Micrographs treatment. (a) treatment. Micrographs show show embryoid bodies bodies embryoid (EB) generated from iPSCsfrom (EB) generated and in vitroand iPSCs in vitro differentiation into differentiation ectodermal into (p11I ectodermal (BIII TUBULIN),mesodermal TUBULIN), (BRACHYURY) mesodermal (BRACHYURY) and and

22

(PDX) endodermal (PDX1) endodermal cell types. Scale celltypes. bars, EB: Scale bars, 100 µm; EB: 100 im; others 10 µm others 10 pm(b) RT-PCR (b) RT-PCR showing showing

expression of expression representativelineage of representative markers lineage andand markers thethe absence of OCT4 absence of OCT4mRNA expression in mRNA expression in cells. U-undifferentiated, differentiating cells. differentiating D-differentiated.(c)(c)Teratomas U-undifferentiated, D-differentiated. Teratomas generated generated in in nude nude micefrom mice iPSCs fromiPSCs (3 independent (3 independent colonies colonies tested) tested) consist consist of tissues of tissues from from all all three three germ layers. germ layers.

5 LeftLeft 5 panel: panel: 1- muscle, 1- muscle, 2- neural 2- neural epithelium; middle middle epithelium; panel: panel: 1- skin, 1- 2- skin, 2- gut epithelium; gut epithelium; right right panel: 1-1-bone, panel: cartilage.Scale bone,2-2-cartilage. bars,2020µm.im. Scalebars, 2022204080

[00851 Figure

[0085] Compound 4. 4.Compound Figure treatment treatment enhanced enhanced iPSgeneration iPS cell in a in cell generation a dose dose dependent dependent

manner. manner.

[0086] Figure

[0086] 5. 5.Chemical Figure Chemical structure Thiazovivin. structureofofThiazovivin.

10 [00871

[0087] Figure Figure 6. Transgene 6. Transgene expression expression and silencing and silencing are independent are independent of compounds of compounds

treatment. treatment.

Figure

[0088] Figure

[0088] Stablyexpanded 7.7.Stably expanded iPS iPS cellcolonies cell colonies generated through compounds generated through compounds treatment exhibitednormal treatment exhibited normal karyotype. karyotype.

[00891 Figure

[0089] Figure 8 Generation 8 Generation of of human human induced induced pluripotent pluripotent cells cells stem stem fromfrom primary primary

15 by by keratinocytes keratinocytes single single OCT4, gene,OCT4, gene, and and small small molecules. molecules. (a) Treatment with with (a) Treatment 0.5 0.5 µM M (PD) PD0325901(PD) PD0325901 0.50.5 andand µMgM A-83-01 A-83-01 (A83) (A83) significantly improved significantlyimproved generation of of generation iPSCs iPSCs

from primary from primary human keratinocytes transduced humankeratinocytes either 4TFs with either transduced with 4TFs (4F, OKSM) oror3TFs (4F,OKSM) 3TFs(3F, (3F, OKS). NHEKs OKS). NHEKswerewere seeded seeded at aatdensity ofof a density 100,000transduced 100,000 cells per transducedcells 10 cm per 10 cmdish. dish. (b) (b) Further chemical Further screens chemicalscreens identified identified PS48, PS48, NaB,NaB, and their and their combination that canthat combination can substantially substantially

enhance 20 enhance 20 reprogramming reprogramming of primary humanhuman of primary keratinocytes keratinocytes transduced transduced 2TFs 2TFs with with (OK).(OK).

NHEKs NHEKs werewere seeded seeded at a density at a density of 100,000 of 100,000 transduced transduced cells percells 10 cmperdish. 10 cm (c) dish. (c) Experimental scheme Experimental for generation schemefor of human generation of human iPSCs fromprimary iPSCsfrom human primaryhuman keratinocytes keratinocytes

singlereprogramming transduced by aa single transduced gene, OCT4. reprogramming gene, KCM, OCT4.KCM, keratinocyte keratinocyte culturemedium; culture medium; human hESCM,human hESCM, ESCESC culture culture media. media. (d) (d) LiveLive immunostaining withwith immunostaining TRA-1-81 TRA-1-81 of of iPSC iPSC 25 colonies 25 colonies weregenerated thatwere that generatedfrom primaryhuman fromprimary human keratinocytestransduced keratinocytes with2TFs/OK transducedwith 2TFs/OK or or picking-upofofcolonies. beforepicking-up ITF/OCT4before 1TF/OCT4 (e) The colonies. (e) established human The established iPSC-OKandand human iPSC-OK iPSC-O iPSC-O

cells express cells typical pluripotency express typical pluripotencymarkers, markers, including including ALP ALP (alkaline (alkaline phosphatase), phosphatase), OCT4, OCT4, SOX2,NANOG, SOX2, NANOG, SSEA-4 SSEA-4 and TRA-1-81. and TRA-1-81. Nuclei Nuclei were stained were stained with with DAPI. DAPI.

[00901 Figure

[0090] Figure 9 In 9 In depth depth characterizations of of characterizations human human iPSC-OK iPSC-OK and iPSC-O and iPSC-O cells. cells. (a) (a) 30 Expression 30 Expression analysis analysis by by RT-PCR RT-PCR of the of the endogenous endogenous pluripotency pluripotency genes and and genes exogenous exogenous OCT4 OCT4 and KLF4.GAPDH and KLF4. GAPDHwas was used used an input asinput as an control. control. (b)(b) Methylation Methylation analysisofofthe analysis the OCT4 OCT4 and and

NANOG NANOG promoters promoters by bisulfategenomic by bisulfate genomic sequencing. sequencing. Open Open circles circles andand closed closed circles circles

23

indicate unmethylated indicate unmethylated andand CpGs CpGs methylated methylated in the in the promoter promoter regions,regions, respectively. respectively. (c) Scatter (c) Scatter

plots global gene comparing global plots comparing expression patterns gene expression cellsand iPSC-O cells betweeniPSC-O patternsbetween andNHEKs, and NHEKs, and

hESCs. of the positionsof Thepositions hESCs. The pluripotency genes the pluripotency genes OCT4, NANOG,andand OCT4, NANOG, SOX2 SOX2 are are shown shown by by arrows. Black arrows. Black lines indicatethethe linesindicate linear linear equivalent equivalent andand twofold twofold changes changes in gene gene expression inexpression 5 levels 5 thethe between levelsbetween samples.(d)(d)Human samples. Human iPSC-OK iPSC-OK and iPSC-O could could and iPSC-O effectively effectively differentiate differentiate

in vitro in vitro into into cells cells in inthe thethree three germ layers, including germ layers, neuralectodermal including neural ectodermal cells cells (pIIItubulin), (BIII tubulin), 2022204080

cells (SMA), mesodermalcells mesodermal andendodermal (SMA), and endodermalcells method.(e)(e)Quantitative usingEBEBmethod. (AFP)using cells(AFP) Quantitative testofofthree PCRtest PCR germ threegerm layer layer markers markers fromfrom differentiated human human differentiated iPSCs iPSCs using EB using EB method: method: ectoderm (PAX6, ectoderm (PAX6,31II TUBULIN),mesoderm ßIII TUBULIN), mesoderm (FOXF, (FOXF1, HAND]) HANDI) and endoderm and endoderm (AFP, (AFP, 10 GATA6). 10 Data Data GATA6). denotes denotes GAPDH-normalized GAPDH-normalized fold changes fold changes relative relative to undifferentiated to undifferentiated parental parental

humaniPSCs. human iPSCs.(f) (f) Human Human iPSC-OK iPSC-OK and and iPSC-O iPSC-O couldcould effectively effectively produce produce fullfull teratoma, teratoma,

whichcontains which cellsininthe differentiatedcells containsdifferentiated thethree germ threegerm layers, layers, in in SCID mice.mice. SCID

[0091] 10 10 Figure

[00911 Figure Generation and and Generation characterizations characterizations of human of human induced induced pluripotent pluripotent

cells from stem cells stem from human human umbilical veinendothelial umbilicalvein cells by endothelialcells gene, OCT4, single gene, by single OCT4, and small andsmall (a) (a) molecules. 15 molecules. 15 Experimental Experimental scheme for for scheme generation of of generation human human iPSCs iPSCs fromfrom HUVECs HUVECs

transduced by transduced by OCT4. HCM, OCT4. HCM, HUVEC HUVEC culture culture medium; medium; hESCM, hESCM, human human ESC ESCmedia. culture culture media. (b) The (b) hiPSC-O establishedhiPSC-O The established cells cells fromfrom HUVECs HUVECs express typical typical pluripotency expresspluripotency markers, markers, including NANOG andand including NANOG SSEA-4. SSEA-4. Nuclei Nuclei were were stained withwith stained DAPI. DAPI. (c) Expression (c) Expression analysis analysis by by RT-PCR RT-PCR ofof theendogenous the endogenouspluripotency GAPDH genes.GAPDH pluripotencygenes. was used as anasinput was used an input control. (d)(d) control. 20 Methylation 20 Methylation analysis of of analysis the OCT4 theOCT4 andand NANOG NANOG promoters promoters by bisulfate by bisulfate genomic genomic sequencing. sequencing.

Opencircles Open andclosed circlesand closed circles indicate circlesindicate unmethylated unmethylated and methylated CpGs in CpGs and methylated in the the promoter promoter regions, (e)hiPSC-O respectively. (e) regions, respectively. hiPSC-Ocells fromfrom cells HUVECs HUVECs could effectively could effectively differentiate differentiate in in vitro into cells vitro into cells in the three in the three germ layers, including germ layers, neuralectodermal includingneural ectodermal cells cells (pIII (BIII tubulin), tubulin),

mesodermal cells (SMA), mesodermalcells andendodermal (SMA), and cells(AFP) endodermalcells method.(f)(f)hiPSC-O usingEBEBmethod. (AFP)using hiPSC-O cells cells

25 couldcould 25 effectively effectively produce produce full teratoma, full teratoma, which contains which contains differentiated cells in cells differentiated in thegerm the three three germ in SCID layers in layers mice. SCID mice.

11 11 Figure

[0092] Figure

[0092] of of Characterization Characterization human human iPSC-O iPSC-O from from cells cells AHEKs. AHEKs. (a) The (a) The established hiPSC-O established hiPSC-O cells cells from from adult adult keratinocytes keratinocytes express express typical typical pluripotency pluripotency markers, markers,

including NANOG, including and and SOX2 NANOG, SOX2 SSEA-4. NucleiNuclei SSEA-4. were stained were stained DAPI.DAPI. with with (b) These (b) These hiPSC-O hiPSC-O

30 cellscells 30 could could effectively effectively differentiate differentiate in vitro in vitro intointo cells cells in the in the three three germgerm layers, layers, including including

neural ectodermal neural cells(BIII ectodermalcells mesodermal tubulin),mesodermal (plil tubulin), cels cell's (SMA), (SMA), and endodermal and endodermal cells cells (AFP) (AFP) using EB using method. EB method.

[0093] 12 12 Figure

[0093] Figure of of Characterization Characterization human human iPSC-O cells cells iPSC-O from from AFDCs. (a) The (a) AFDCs. The established hiPSC-O established hiPSC-O cells cells from from amniotic fluid fluid amniotic derived derived cells cells express express typical typical pluripotency pluripotency

24

markers, including markers, including NANOG, SOX2 NANOG, SOX2 and and SSEA-4. SSEA-4. Nuclei Nuclei were were stained with with stained DAPI.DAPI. (b) These (b) These

hiPSC-O hiPSC-O cellscould cells could effectively effectively differentiate differentiate in in vitro vitro into into cellsininthe cells thethree threegerm germ layers, layers,

neuralectodermal includingneural including ectodermal cells cells (BIII tubulin),mesodermal (pIIItubulin), cellscells mesodermal (SMA), (SMA), and endodermal and endodermal

cells(AFP) cells using EB (AFP) using EB method. method.

5 [0094] 5 [0094] Figure Figure 13 Additional hiPSC hiPSC 13 Additional cell lines cell lines express express typical typical pluripotency pluripotency markers. markers.

2022204080 otherestablished Theother The establishedhiPSC-O cell cell hiPSC-O lines lines express express typical typical pluripotency pluripotency markers, markers, including including

NANOG NANOG and and SSEA-4. SSEA-4. Nuclei Nuclei were were stained with with stained DAPI. DAPI.

[00951 Figure

[0095] 14 14 Figure Feeder Feeder free free cultureofofhiPSC culture hiPSC celllines. cell lines. hiPSCs weresplit hiPSCswere split onto onto Matrigel/ECM-coatedplates Matrigel/ECM-coated chemically defined in chemically plates in defined hESC mediumasaspreviously hESC medium reported. previouslyreported. 10 10 These These hiPSCs hiPSCs could could be maintained and and be maintained expanded in ainfeeder-free expanded environment.ICCICC a feeder-freeenvironment. showed showed

the expression of the expression ofpluripiotency pluripiotencymarkers, markers, OCT4 and OCT4 and SSEA4. Nucleiwere SSEA4. Nuclei stained with werestained DAPI. with DAPI.

[0096] 15 15 Figure

[0096] Figure Genotyping Genotyping of hiPSCs. of hiPSCs. RT-PCR RT-PCR analysis using using analysis genomic genomic DNA DNA shows shows that only that onlyOCT4 integrated ininthe transgene integrated OCT4 transgene genome of thegenome ofhiPSC-O hiPSC-O lines (hiPSC-O#1, hiPSC lines (hiPSC-O#1, hiPSC-

O#3, hiPSC-O#26 hiPSC-O#21,hiPSC-O#26 O#3, hiPSC-O#21, and and hiPSC-O#31). NHEKsNHEKs hiPSC-O#31). (a) and(a) and HUVECs HUVECs (b) used (b) as used as 15 negative 15 negative whilewhile controls, controls, vectors vectors used used as as positive positive controls. controls.

16 16 Figure

[0097] Figure

[0097] of of Integration Integration the OCT4 theOCT4 transgene transgene in hiPSCs. in hiPSCs. Genomic Genomic DNA (10 µg) (10 DNA g) digested with were digested were with EcoRI EcoRI and hybridized with and hybridized the OCT4 with the cDNA OCT4 cDNA probe probe (an(an EcoRI/Spel EcoRI/Spel

fragmentofofpSin-EF2-OCT4-Pur). fragment pSin-EF2-OCT4-Pur). Multiple Multiple transgenic transgenic integrations integrations were were detected. detected.

[0098] Figure

[0098] Figure 17 17 for for Karyotyping Karyotyping hiPSC hiPSC cell cell lines.Metaphase lines. Metaphase spread spread of hiPSC-O#1 of hiPSC-O#1 (a) (a) 20 andand 20 hiPSC-O#21 hiPSC-O#21 (b) show (b) show normal normal karyotype afterafter karyotype passage passage 15. 15.

DETAILED DESCRIPTION DETAILED DESCRIPTION

I. Introduction I. Introduction

[00991 TheThe

[0099] present present invention invention is based is based on theon the surprising surprising discovery discovery that a combination that a combination of an of an 25 ALK5ALK5 25 inhibitor, inhibitor, a MEK a MEK inhibitor, inhibitor, andand a ROCK a ROCK inhibitor inhibitor greatly greatly improves improves efficiencyofof efficiency

pluripotencyin innon-pluripotent induction ofofpluripotency induction non-pluripotent mammalian mammalian cells transformed with fourwith cells transformed four factors. Accordingly, transcription factors. transcription Accordingly,thethe present present invention invention provides provides for methods for methods of inducing of inducing

pluripotency ininnon-pluripotent pluripotency non-pluripotent mammalian mammalian cells wherein cells wherein the comprises the method contacting contacting method comprises the cellswith non-pluripotentcells the non-pluripotent leastaaTGFß withatatleast TGFreceptor/ALK5 receptor/ALK5 inhibitor, inhibitor, preferably preferably in in 30 withwith combination combination a MEK/ERK a MEK/ERK pathway pathway inhibitor, inhibitor, and in in particular andparticular embodiments, embodiments, a a Rho Rho GTPase/ROCK GTPase/ROCK inhibitor. inhibitor.

25

H. TGF/Jreceptor/ALK5 II. TGFBreceptor/ALK5 inhibitors inhibitors

[01001Activin

[0100] Activin receptor-like receptor-like kinase kinase 5 (ALK-5) 5 (ALK-5) is the principal is the principal TGFPthat TGF receptor receptor that mediates mediates cellular responses cellular responsestoto TGF-ps TGF-ßs(Massague (Massague J. J. Annu Annu Rev Rev Biochem 67:753-791(1998); Biochem 67:753-791 (1998);Massague Massague J, Chen J, YG.Genes Chen YG. Genes Dev Dev 14:627-644 14:627-644 (2000); (2000); Franzen Franzen P,Cell P, et al.. et al.. Cell 75:681-692 75:681-692 (1993)). (1993)). 55 Upon Upon ligand ligand binding, binding, constitutively active constitutively active TpRII kinase phosphorylates TBRII kinase phosphorylates ALK-5 which,inin ALK-5 which,

turn, activates turn, activates the the downstream signal downstream signal transduction transduction cascades. cascades. ALK-5-activated ALK-5-activated Smad2 andSmad2 and 2022204080

Smad3phosphorylation Smad3 phosphorylationisis the the most prominent pathway most prominent pathway (Massague (MassagueJ,J,Chen ChenYG. YG.Genes Genes Dev Dev

14:627-644(2000)). 14:627-644 (2000)). OnceOnce activated, activated, Smad2/3 Smad2/3 associates associates with with Smad4 andSmad4 and translocates translocates to the to the nucleus, where nucleus, wherethethecomplex complex transcriptionally transcriptionally regulates regulates target target gene gene expression. expression.

10 10 [01011

[0101] TGFp receptor TGF receptor (i.e. ALK5) (i.e. ALK5) inhibitors inhibitors can include can include antibodies antibodies to, to, dominant dominant negative negative

variants of, variants of, and siRNA,microRNA, and siRNA, microRNA, antisense antisense nucleicnucleic acids, acids, andpolynucleotides and other other polynucleotides that that suppress expression suppress expression of, of,TGFp receptors (e.g., TGF receptors (e.g., ALK5). ExemplaryTGFß ALK5). Exemplary TGFP receptor/ALK5 receptor/ALK5

inhibitors include, inhibitors include, but but are are not not limited limitedto, to, SB431542 SB431542(see,(see, e.g., e.g., Inman, Inman, et al., et al., Molecular Molecular

Pharmacology 62(1):65-74(2002)), Pharmacology 62(1):65-74 (2002)), A-83-01, A-83-01, also also known knownasas3-(6-Methyl-2-pyridinyl)-N- 3-(6-Methyl-2-pyridinyl)-N 15 15 phenyl-4-(4-quinolinyl)-IH-p phenyl-4-(4-quinolinyl)-1H-p yrazole--carbothioamide yrazole-1-carbothioamide (see, e.g., (see, Tojo,e.g., Tojo, et al., et al., Cancer Cancer Science96(11):791-800 Science 96(11):791-800 (2005), (2005), and commercially and commercially available available from, from, e.g., e.g., Toicris Toicris Bioscience); Bioscience);

2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a/BIO 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a/BIO (see, e.g.,(see, e.g.,

Dalton, etet al., Dalton, al., W02008/094597, herein WO2008/094597, herein incorporated incorporated by reference), by reference), BMP4 BMP4 (see, (see, Dalton, Dalton, supra), GW788388 supra), GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-

20 pyran-4-yl)benzamide) 20 pyran-4-yl)benzamide) (see,Gellibert, (see, e.g., e.g., Gellibert, et al.,etJournal al., Journal ofMedicinal of Medicinal Chemistry Chemistry

49(7):2210-2221 49(7):2210-2221 (2006)), (2006)), SM16SM16 (see, (see, e.g., e.g., Suzuki, Suzuki, et Cancer et al., al., Cancer Research Research 67(5):2351-2359 67(5):2351-2359

(2007)), IN-1 (2007)), 130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-IH-imidazol-2 IN-1130 (3-(5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1-imidazol-2-

yl)methyl)benzamide) yl)methyl)benzamide) (see, (see, e.g., e.g., Kim, Kim, et al., et al., Xenobiotica Xenobiotica 38(3):325-339 38(3):325-339 (2008)), (2008)), GW6604 GW6604 (2-phenyl-4-(3-pyridin-2-yl-IH-pyrazol-4-yl)pyridine) (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., (see, e.g., de Gouville, de Gouville, et Drug et al., al., Drug 25 NewsNews 25 Perspective Perspective 19(2):85-90 19(2):85-90 (2006)), (2006)), SB-505124 SB-505124 (2-(5-benzo[1,3]dioxol-5-y-2-tert-butyl (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-

3H-imidazol-4-y)-6-methylpyridine 3H-imidazol-4-yl)-6-methylpyridine hydrochloride) hydrochloride) (see, DaCosta, (see, e.g., e.g., DaCosta, et al., et al., Molecular Molecular

Pharmacology Pharmacology 65(3):744-752 65(3):744-752 (2004)) (2004)) and pyrimidine and pyrimidine derivatives derivatives (see, (see, e.g., e.g., those thoseinlisted listed in Stiefl, etetal., Stiefl, al.,W02008/006583, herein WO2008/006583, herein incorporated incorporated by reference). by reference). Further, Further, while while "an "an ALK5 ALK5 inhibitor" is inhibitor" is not not intended to encompass intended to encompass non-specific non-specific kinase kinase inhibitors, inhibitors, an "ALK5 an "ALK5 inhibitor" inhibitor"

30 30 should should be be understood understood to to encompass encompass inhibitorsthat inhibitors thatinhibit inhibit ALK4 and/orALK7 ALK4 and/or ALK7in in additiontoto addition

ALK5, ALK5, as,as, such such forfor example, example, SB-431542 SB-431542 e.g., Inman, (see, Inman, (see, e.g., et al., et J,al., Mol.J, Phamacol. Mol. Phamacol. 62(1): 62(1):

65-74(2002). 65-74 (2002).

[01021 In view

[0102] In view of the of the datadata herein herein showing showing the effect the effect of inhibiting of inhibiting ALK5, ALK5, it it is believed is believed that that inhibition of inhibition of the the TGFß/activin TGFp/activin pathway pathway will will have have similar similar effects. effects. Thus, Thus, any inhibitor any inhibitor (e.g., (e.g.,

26

upstreamorordownstream) upstream downstream) of TGFß/activin of the pathwaypathway the TGFp/activin can in can be used used in combination becombination with, or with, or instead of, ALK5 instead of, ALK5 inhibitors as as inhibitors described described in each in each paragraph paragraph herein. herein. Exemplary Exemplary TGFp/activin TGFß/activin

pathwayinhibitors pathway butbut include inhibitorsinclude areare notnot limited to: to: limited TGFp TGFß receptor receptor inhibitors, inhibitors, inhibitors inhibitors of of SMAD SMAD 2/32/3 phosphorylation,inhibitors phosphorylation, the interaction of the inhibitors of interactionofof SMAD 2/3 SMAD 2/3 and SMAD 4,4,and and SMAD and 5 activators/agonists 5 of SMAD activators/agonists of SMAD 6 and 6 and SMAD SMAD 7. Furthermore, 7. Furthermore, the categorizations the categorizations described described

aremerely beloware below merelyforfor organizational organizational purposes purposes andofone and one of skill skill in thein art art would the would know know that that 2022204080

compoundscan compounds affect one canaffect or more one or more points pathway, and within aa pathway, points within thuscompounds and thus may compounds may

function in more function in thanoneone morethan thethe of of defined defined categories. categories.

[0103] TGFßTGFp

[0103] receptor receptor inhibitors inhibitors can include can include antibodies antibodies to, dominant to, dominant negative ofvariants negative variants of 10 andand 10 siRNA siRNA or antisense or antisense nucleic nucleic acids thattarget acidsthat target TGFO TGFß receptors. Specific examples receptors. Specific of examples of

inhibitors include inhibitors butare include but are not limitedtotoSU5416; notlimited SU5416; 2-(5-benzo[1,3]dioxo-5-y-2-tert-butyl-3H 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-

hydrochloride (SB-505124); imidazol-4-yl)-6-methylpyridine hydrochloride imidazol-4-yl)-6-methylpyridine (SB-505124); lerdelimumb (CAT-152); lerdelimumb (CAT-152); metelimumab (CAT-192); metelimumab (CAT-192); GC-1008; GC-1008; IDI1; ID11; AP-12009; AP-12009; AP-11014; AP-11014; LY550410; LY550410; LY580276; LY580276;

LY2109761; LY364947;LY2109761; LY364947; SB-505124; SB-505124; SB-431542; SB-431542; SD-208; SM16; SM16; SD-208; NPC-30345; Ki26894; Ki26894; NPC-30345; 15 SB-203580; 15 SB-203580; SD-093; SD-093; Gleevec; Gleevec; 3,5,7,2',4'-pentahydroxyflavone 3,5,7,2',4'-pentahydroxyflavone (Morin); (Morin); activin-MI08A; activin-M108A;

TBR2-Fc; solubleTBR2-Fc; P144;soluble P144; and antisense and antisense transfected transfected tumor tumor cells that that target cellstarget TGF receptors. TGFß receptors.

(See, e.g., (See, e.g., Wrzesinski, et al., Wrzesinski, et al., Clinical Cancer Clinical Cancer Research Research 13(18):5262-5270 13(18):5262-5270 (2007);(2007); Kaminska, Kaminska,

et al., et Acta Biochimica al.,Acta Polonica Biochimica Polonica 52(2):329-337 52(2):329-337 (2005); (2005); and Chang, and Chang, et al., et al., Frontiers Frontiers in in 12:4393-4401 Bioscience12:4393-4401 Bioscience (2007). (2007). In addition, In addition, the inventors the inventors have found the that found havethat TGFßthe TGF3 20 inhibitors 20 and and BMP-4 inhibitorsBMP-4 BMP-7 have have BMP-7 similar similar cellular cellular reprogramming reprogramming effects as as effects thethe ALK5 ALK5

the examples, describedinin the inhibitor described inhibitor thereby examples,thereby providing providing further further evidence evidence that TGFß TGFP inhibitors that inhibitors can be used for can be for reprogramming reprogramming (e.g., combinationwith (e.g., inincombination witha MEK/ERK a MEK/ERK pathway and inhibitor and pathwayinhibitor a Rho a GTPase/ROCK Rho GTPase/ROCK Exemplary inhibitor).Exemplary inhibitor). human human BMP-4BMP-4 and BMP-7 and BMP-7 protein protein sequences sequences

are set are set forth forth in, forexample, in, for example, US PatentNo.No. USPatent 7,405,192. 7,405,192.

25 [0104] 25 [0104] Inhibitors Inhibitors of SMAD of SMAD 2/3 phosphorylation 2/3 phosphorylation can include can include antibodies antibodies to, dominant to, dominant

variantsofofand negative variants negative antisense andantisense nucleic nucleic acids acids that that target target SMAD2 SMAD2 or SMAD3. or SMAD3. Specific Specific examples inhibitors include of inhibitors examples of PD169316; SB203580; includePD169316; SB203580; SB-431542; LY364947; SB-431542;LY364947; A77-01; A77-01; and and 3,5,7,2',4'-pentahydroxyflavone (Morin). 3,5,7,2',4'-pentahydroxyflavone (Morin). e.g.,e.g., (See,(See, Wrzesinski, Wrzesinski, supra;supra; Kaminska, Kaminska, supra; supra;

Shimanuki,et etal., Shimanuki, al.,Oncogene Oncogene26:3311-3320 (2007);(2007); 26:3311-3320 and Kataoka, and Kataoka, et al., EP992360, et al., EP1992360,

incorporated 30 incorporated 30 hereinby by herein reference.) reference.)

[0105] Inhibitors

[0105] Inhibitors of the of the interaction interaction of SMAD of SMAD 2/3 and2/3 andcan smad4 smad4 canantibodies include include antibodies to, to, dominantnegative dominant negative variants variants of and of and antisense antisense nucleic nucleic that that acidsacids target target SMAD2, SMAD2, SMAD3 SMAD3 and/or and/or smad4. examplesof Specific examples smad4. Specific inhibitors of ofinhibitors ofthe theinteraction interaction of SMAD2/3 of SMAD 2/3and andSMAD4 include SMAD4 include

27

but are but are not not limited limited to to Trx-SARA, Trx-SARA, Trx-xFoxHlb Trx-xFoxH1b and Trx-Lefl. and Trx-Lefl. (See, (See, e.g., Cui,e.g., Cui, et al., et al., Oncogene Oncogene (2005)(2005) 24:3864-3874 24:3864-3874 and Zhao, al., et and etZhao, al., Molecular Molecular Biology Biology of of the the Cell, Cell, 17:3819- 17:3819 3831(2006).) 3831 (2006).)

[0106]Activators/agonists

[0106] Activators/agonistsof of 6 and 6 SMAD SMAD include 7 and7 SMAD SMAD include but are notbut are not limited to limited to antibodies antibodies 5 to,to,dominant 5 dominant variantsofofand negativevariants negative nucleic acids antisense nucleic and antisense acidsthat target that target SMAD66ororSMAD SMAD SMAD

2022204080 Specific examples 7. Specific 7. examplesof of inhibitors inhibitors include include but but are are not not limited limited to smad7-as to smad7-as PTO- PTO oligonucleotides. See,e.g., oligonucleotides.See, Miyazono, e.g.,Miyazono, et al., et al., US6534476, US6534476, and Steinbrecher, and Steinbrecher, et al., et al., US2005119203,both US2005119203, incorporatedherein bothincorporated hereinby reference. byreference.

Those

[01071 Those

[0107] of skill of skill willwill appreciate appreciate the the that that concentration concentration of theof the receptor/ALK5 TGFß TGF receptor/ALK5 10 10 willwill inhibitor inhibitor depend depend on which on which specific specific inhibitor inhibitor is Generally, is used. used. Generally, the concentration the concentration of a of a TGFP TGF receptor/ALK5 receptor/ALK5 inhibitor inhibitor in aculture in a cell cell culture will will be in be the in the range range of IC20-IC100 of IC20-IC100 (i.e., (i.e., concentrationsininwhich concentrations which20%20% inhibition inhibition to 100% to 100% inhibition inhibition in is in cells cells is achieved. achieved. For example, For example,

would SB432542would SB432542 be be usedatat0.5-10 used gM,optimally 0.5-10 µM, 1-5µM.M.In Incertain around1-5 optimallyaround embodiments,a certainembodiments, a combinationof of combination twotwo or or more more different TGFßTGFP different receptor/ALK5 inhibitorsinhibitors receptor/ALK5 can can be used. be used.

HI. MEK/ERK 15 III. 15 MEK/ERKpathway inhibitors pathway inhibitors

[01081 TheThe

[0108] MEK/ERK MEK/ERK pathway pathway refersrefers to theto MEK the MEK and ERK ERK serine/threonine andserine/threonine kinases kinases that that partofof makeupuppart make a a transduction signaltransduction signal pathway. pathway. Generally, Generally, activated activated Ras activates Ras activates the protein the protein

ofRAF activity of kinase activity kinase RAF kinase. kinase. RAFRAF kinase kinase phosphorylates phosphorylates and activates and activates MEK, MEK, which which in turn in turn and activates phosphorylates and phosphorylates mitogen-activatedprotein activatesa amitogen-activated kinase protein kinase (MAPK).MAPK (MAPK). was MAPK was

originally 20 originally 20 called called "extracellular "extracellular signal-regulated signal-regulated kinases" (ERKs) (ERKs) kinases" and microtubule-associated and microtubule-associated

protein kinase protein kinase(MAPK). Thus,"ERK" (MAPK). Thus, "ERK"andand "MAPK" "MAPK" are used are used synonymously. synonymously.

MEK/ERK

[01091 MEK/ERK

[0109] pathway pathway inhibitors referrefer inhibitors to inhibitorsofofeither to inhibitors MEKororERK either MEK ERK that that areare part of part ofthe theRaf/MEK/ERK pathway.Because Raf/MEK/ERK pathway. thethe Because inventors found havefound inventorshave thatMEK that MEK inhibitors inhibitors

are effective are in improving effective in improvinginduction of of induction iPSCs, and and iPSCs, because because MEK directly MEK directly controls controls ERK ERK 25 activity, 25 activity, it isbelieved it is believed that that MEKMEK inhibitors inhibitors as described for thefor as described the present present invention, invention, can be can be replaced withananERK replaced with ERK inhibitor inhibitor as desired. as desired.

[0110] Inhibitors

[0110] of MEK Inhibitors of (i.e., MEKI MEK(i.e., (alsoknown MEK1 (also knownas as mitogen-activated protein kinase mitogen-activatedprotein kinase and/orMEK2 kinase 1)1) and/or kinase MEK2(also(also known known as mitogen-activated as mitogen-activated proteinkinase protein kinase 2))kinase kinase can 2)) can include antibodies include antibodiesto, dominant to, dominantnegative negativevariants of, of, variants and and siRNA andand siRNA siRNA, siRNA,microRNA, microRNA,

30 antisense 30 antisense nucleic nucleic acids, acids, and other and other polynucleotides polynucleotides that suppress that suppress expression of, MEK. of, MEK. expression Specific examples Specific examplesof of MEKMEK inhibitors inhibitors include, include, butnot but are arelimited not limited to, PD0325901, to, PD0325901, (see, (see, e.g., e.g., Rinehart, etet al., Rinehart, al., Journal ofClinical Journal of ClinicalOncology Oncology22: 22: 4456-4462 4456-4462 (2004)), (2004)), PD98059 PD98059 (available, (available,

e.g., from e.g., Cell Signaling from Cell Technology), SignalingTechnology), U0126 U0126 (available, (available, for example, for example, from from Cell Cell Signaling Signaling

28

327 327 Technology),SL SL Technology), (available, (available, e.g., e.g., fromfrom Sigma-Aldrich), Sigma-Aldrich), ARRY-162 e.g., from e.g., (available,(available, ARRY-162 from Array Biopharma), ArrayBiopharma), PD184161 PD184161 (see, e.g., e.g., Klein, (see, Klein, et al., al., Neoplasia et Neoplasia 8:1 - 8 (2006)), 8:1 8 (2006)), PD184352 PD184352 (CI-1040)(see, (CI-1040) al., The Mattingly,etetal., e.g., Mattingly, (see,e.g., The Journal JournalofofPharmacology and Experimental Pharmacology and Experimental

Therapeutics Therapeutics 316:456-465 316:456-465 (2006)), (2006)), sunitinib (see, (see, sunitinib e.g.,e.g., Voss,Voss, et al., et al., US2008004287 US2008004287

incorporated 5 incorporated 5 herein herein by reference), by reference), sorafenib sorafenib (see, Voss Voss supra), (see,supra), Vandetanib (see, Voss(see, Vandetanib Voss supra), supra), (see,e.g., pazopanib(see, pazopanib Vosssupra), e.g.,Voss supra), Axitinib Axitinib (see, (see, Voss Voss supra) supra) and PTK787 and PTK787 (see, (see, Voss Voss supra). supra). 2022204080

[01111 Currently,

[0111] Currently, several several MEK inhibitors MEK inhibitors are undergoing clinical clinical are undergoing trial evaluations. trial evaluations. CI- CI 1040has 1040 hasbeen beenevaluate evaluate in in Phase Phase I and I and II clinical II clinical trialsforforcancer trials cancer (see,e.g., (see, e.g.,Rinehart, Rinehart,etetal., al., Journalof Journal Oncology 22(22):4456-4462 ClinicalOncology of Clinical 22(22):4456-4462 (2004)). MEKinhibitors Other MEK (2004)). Other being inhibitors being 10 evaluated 10 evaluated in clinical in clinical trials trials include include PD184352 PD184352 (see, English, (see, e.g., al., Trends et al.,etTrends e.g., English, in in Pharmaceutical Sciences Pharmaceutical Sciences 23(1):40-45 23(1):40-45 (2002)), (2002)), BAY (see, BAY 43-9006 43-9006 (see, e.g., e.g., Chow, Chow, et al., et al.,

(Communications Cytometry(Communications Cytometry ininClinical ClinicalCytometry) (2001)), PD-325901 46:72-78(2001)), Cytometry)46:72-78 (also PD-325901(also PD0325901), GSKI120212, PD0325901), GSK1120212, ARRY-438162, RDEA119, ARRY-438162,RDEA119, AZD6244 AZD6244 (also (also ARRY-142886 ARRY-142886 or or RO5126766, ARRY-886),RO5126766, ARRY-886), XL518 XL518 and AZD8330 and AZD8330 (also ARRY-704). (also ARRY-704). (See, (See, e.g., e.g., information information

15 fromfrom 15 the National the National Institutes Institutes of Health of Health located on the on located the Wide World Web Wide World Web at clinicaltrials.gov at clinicaltrials.gov as as as information well as well informationfrom thethe from Nation Nation Cancer Cancer Institute Institute located located on the the World onWorld Wide Wide Web at Web at cancer.gov/clinicaltrials. cancer.gov/clinicaltrials

[0112] Exemplary

[0112] ERKERK Exemplary (i.e., (i.e., ERKI ERK1 (also (also known known as MAPK3) and/orand/or as MAPK3) ERK2 (also (also known ERK2known as as MAPK1)) MAPK1)) inhibitors inhibitors include include PD98059 PD98059 (see,Zhu, (see, e.g., al.,et e.g.,etZhu, al., Oncogene Oncogene 23:4984-4992 23:4984-4992

(2004)), 20 (2004)), 20 U0126 U0126 (see, (see, Zhu, Zhu, supra), supra), FRI80204 FR180204 (see, (see, e.g.,Ohori, e.g., NewsPerspective DrugNews Ohori, Drug Perspective 21(5):245-250(2008)), 21(5):245-250 (2008)), sunitinib sunitinib (see, (see, e.g.,US2008004287 e.g., US2008004287 incorporated incorporated herein herein by by reference), reference),

sorafenib, Vandetanib, pazopanib, sorafenib, Vandetanib, Axitiniband pazopanib, Axitinib andPTK787. PTK787.

[01131 Those

[0113] of of Those skill will skill appreciatethat will appreciate thatthethe concentration of the concentration of the MEK/ERK pathway MEK/ERK pathway

inhibitor depend ononwhich will depend inhibitor will which specific specific inhibitor inhibitor is used. is used. In particular In particular embodiments, embodiments, a a 25 combination 25 combination of two of two or more or more different MEK/ERK differentMEK/ERK pathway pathway inhibitors inhibitors can be be used. canused.

IV. RhoRho IV. GTPase/ROCK GTPase/ROCK Inhibitors Inhibitors

[0114] The present invention

[0114] The present provides provides invention for uses uses forand compositions comprisingcomprising and compositions inhibitors inhibitors of of the Rho-GTPase/ROCK the pathway. Rho-GTPase/ROCK pathway. The pathway The pathway includes includes the downstream the downstream protein protein MyosinMyosin II, II,

is further which is which downstream of ROCK furtherdownstream (RhoROCK-Myosin ROCK (Rho-ROCK-Myosin Il forms II forms the pathway/axis). the pathway/axis).

30 Thus,Thus, 30 oneuse one can all or useorany canany of all a Rho Rho GTPase of aGTPase inhibitor, inhibitor, a ROCK inhibitor, II a a Myosin or a ROCK orinhibitor, Myosin II to achieve inhibitor to inhibitor effects described theeffects achieve the herein.Those describedherein. Those of skill of skill willwill appreciate thatthat appreciate the the of the concentration of concentration theRho-GTPase/ROCK pathway Rho-GTPase/ROCK pathway inhibitor inhibitor will dependononwhich willdepend which specific specific

29

inhibitor is used. inhibitor is In additional used. In embodiments, additional embodiments, a combination ortwo of twoof a combination more different moreordifferent Rho- Rho GTPase/ROCK pathway GTPase/ROCK pathway inhibitors inhibitors cancan be be used. used.

Any

[01151 Any

[0115] RhoRho GTPase GTPase should should be effective in in be effective the methods themethods and and compositions of of compositions the the

InhibitorsofofRho invention. Inhibitors invention. Rho GTPase GTPase can include can include antibodies antibodies that bind, negative negative bind, dominant that dominant 5 variants 5 of, of, variants and and siRNA, siRNA, microRNA, antisense antisense microRNA, nucleic acids, and acids, nucleic and other polynucleotides other polynucleotides that that

2022204080 targetRho target Rho GTPase. exemplaryRhoRho Anexemplary GTPase. An GTPase GTPase inhibitor is isClostridium inhibitor botulinumC3C3toxin. Clostridiumbotulinum toxin.

[0116] AnyAny

[0116] Myosin Myosin II inhibitor shouldshould II inhibitor be effective be effective in the methods in the methods and compositions and compositions of the of the InhibitorsofofMyosin invention. Inhibitors invention. MyosinII II cancan include include antibodies that that antibodies bind,bind, dominant dominant negative negative

of, and variants of, variants microRNA, siRNA,microRNA, and siRNA, antisense antisense acids, acids, nucleicnucleic and other other polynucleotides andpolynucleotides that that target 10 target 10 II. An11.exemplary Myosin Myosin An exemplary Myosin Myosin II II inhibitor inhibitor is blebbistatin. is blebbistatin. The inventors inventors The have have that blebbistatin foundthat found canbebesubstituted blebbistatincan SB431542 substitutedforforSB431542 (an ALK5 ALK5 inhibitor), (an inhibitor), albeita albeit with with a reducedeffect, reduced mixturesandand the mixtures effect, inin the methods methods described the example in theinexample described section. section. Other Other inhibitors include inhibitors but are include but limitedtotothose notlimited are not described thosedescribed in in US US Patent No. No. Patent 7,585,844. 7,585,844.

[0117] "ROCK"

[01171 "ROCK" used herein herein to used refers refers to a serine/threonine a serine/threonine kinase that actsthat kinase acts downstream downstream of of 15 Rho. 15 ROCKROCK Rho. I (also I (also referred referred to as ROKROK to as P or p160ROCK) ß or p160ROCK) andIIROCK and ROCK to as to (also referred (alsoIIreferred as ROKora Rho ROK or Rho kinase) kinase) are regulated are both both regulated by See by RhoA. RhoA. e.g., See e.g.,K.Riento, Riento, K. andA.J., and Ridley, Ridley, A.J., Nat. Mol.Cell. Rev. Mol. Nat. Rev. Biol.,4,4, 446-456 Cell.Biol., 446-456 (2003). (2003). A "ROCK A "ROCK inhibitor" inhibitor" refers refers to agents agents that to that inhibit inhibit both or either both or either of of the the ROCKs. Inhibitors ROCKs. Inhibitors of ROCK of ROCK can include can include antibodies antibodies that bind, that bind,

dominant negative dominantnegative of,of, variants variants andand siRNA, siRNA, microRNA, antisenseantisense microRNA, nucleic acids, other and andacids, nucleic other polynucleotides 20 polynucleotides 20 thattarget that ROCK. target ROCK. Some Some exemplary ROCK ROCK exemplary inhibitors inhibitors include, include, but not but are are not limited to, those limited to, describedininInternational those described InternationalPublication Publication Nos.: Nos.: W098/06433, WO98/06433, WOOO/78351, WO00/78351,

WO01/17562, WO02/076976,WO02/076977, WO01/17562, WO02/076976, W02003/062227, W002/076977,WO2003/062227, W02003/059913, WO2003/059913,

W02003/062225, WO2003/062225, WO2002/076976, W02004/039796, W02002/076976,WO2004/039796, WO03/082808, WO03/082808, WO05/035506, WO05/035506,

W005/074643 WO05/074643 andand United United StatesPatent States PatentApplication ApplicationNos.: Nos.:2005/0209261, 2005/0209261, 2005/0192304, 2005/0192304,

25 2004/0014755, 25 2004/0002508, 2004/0002507, 2004/0014755,2004/0002508, 2003/0125344 and 2004/0002507, 2003/0125344 2003/0087919.ROCK and 2003/0087919. ROCK

include, for inhibitors include, inhibitors (+)-(R)-trans-4-(I-aminoethyl)-N-(4 example,(+)-(R)-trans-4-(1-aminoethyl)-N-(4- for example, piridyl)cyclohexanecarboxamide dihydrochloride, or piridyl)cyclohexanecarboxamide dihydrochloride, or Wf536; 4-[(1R)-1-aminoethyl]-N-(4 Wf536; 4-[(1R)-1-aminoethyl]-N-(4-

monohydrochlorideororFasudil; piridyl)benzamide monohydrochloride piridyl)benzamide 5-(hexahydro-IH-1,4-diazepin-1 Fasudil; 5-(hexahydro-1H-1,4-diazepin-1- ylsulfonyl)isoquinoline hydrochlorideororCompound ylsulfonyl)isoquinoline hydrochloride 1; 4-[(trans-4-aminocyclohexyl)amino] Compound 1; 4-[(trans-4-aminocyclohexyl)amino]-

30 2,5-difluorobenzamide 30 2,5-difluorobenzamide or Compound or Compound 2; 4-[(trans-4-aminocyclohexyl)amino]-5-chloro-2 2; 4-[(trans-4-aminocyclohexyl)amino]-5-chloro-2-

or Compound fluorobenzamide or fluorobenzamide Compound 3; 3; 2-[4-(H-indazol-5-yl)phenyl]-2-propanamine 2-[4-(1H-indazol-5-yl)phenyl]-2-propanamine

dihydrochloride or dihydrochloride Compound 4;4;N-(3-methoxybenzyl)-4-(4-piridyl)benzamide, or Compound Y-27632 N-(3-methoxybenzyl)-4-(4-piridyl)benzamide, Y-27632 Ishizaki et e.g.,Ishizaki (see, e.g., (see, etal., al.,Mol. Mol.Pharmacol. Pharmacol. 57, 976-983 57,976-983 (2000); (2000); Narumiya Narumiya et al., al., Methods etMethods Enzymol. 325,273-284 Enzymol.325,273-284 (2000)), (2000)), Fasudil Fasudil (also (also referred to as to referred as HAI HA1077) 077) (for (for example, example, refer to refer to

30

al., Nature Uenataetetal., Uenata 990-994 389:990-994 Nature 389: (1997)), (1997)), sc-3536 sc-3536 e.g.,e.g., (see, (see, Darenfed, H., etH., Darenfed, al.etCell al. Cell Motil. Motil.

64:97-109, Cytoskeleton.64: Cytoskeleton. 97-109, 2007), 2007), H-1152 H-1152 (for example, (for example, refer refer to to Sasaki Sasaki al., Pharmacol. et al.,etPharmacol.

93: 225-232 Ther. 93: Ther. (2002)), 225-232(2002)), Wf-536 (for (for Wf-536 example, refer refer example, to Nakajima et al., et to Nakajima al., Cancer Cancer Chemother Chemother

Pharmacol. 52(4): Pharmacol. 52(4): 319-324 319-324 (2003)), (2003)), Y-30141 Y-30141 (described (described in USNo.Patent in US Patent No. 5,478,838) 5,478,838) and and 55 derivatives derivatives thereof, thereof, and and antisense antisense nucleic nucleic acidROCK, acid for for ROCK, RNA interference RNA interference inducing inducing nucleic nucleic acid (for acid (for example, example,siRNA), siRNA), competitive competitive peptides, peptides, antagonist antagonist peptides, peptides, inhibitory inhibitory antibodies, antibodies, 2022204080

antibody-ScFV antibody-ScFV fragments, fragments, dominant dominant negative negative variants variants and expression and expression vectors vectors thereof. thereof.

[0118] The

[0118] The above above may maybe compounds compounds be made made as acid as acid addition addition salts salts with with pharmaceutically pharmaceutically

acidsor ororganic inorganicacids acceptableinorganic acceptable organic acids, as as acids, required. required. Examples of the of Examples theaddition acid acid addition salts salts 10 include 10 include saltssalts withwith mineral mineral acids acids such such as as hydrochloric hydrochloric acid, hydrobromic acid, hydrobromic acid, acid, acid, sulfuric sulfuric acid, phosphoric acidandand phosphoricacid thethe like;salts like; withorganic saltswith organic carboxylic carboxylic acids such such acids as formic as formic aceticacetic acid, acid,

acid, fumaric acid, acid, maleic fumaricacid, acid,oxalic maleicacid, acid,citric oxalicacid, acid, malic citricacid, acid,tartaric malicacid, acid, aspartic tartaric acid, acid aspartic acid and acid;andand glutamicacid; and glutamic salts withsulfonic saltswith sulfonic acids acids such such as methanesulfonic as methanesulfonic acid, acid, benzenesulfonicacid, benzenesulfonic acid,p-toluenesulfonic p-toluenesulfonic acid, acid, hydroxybenzenesulfonic hydroxybenzenesulfonic acid, acid, dihydroxybenzene 15 dihydroxybenzene 15 sulfonic sulfonic acid andand acid thethe like. like.

[0119] The

[0119] The compounds acidacid and and compounds addition addition saltsthereof salts maybe thereof may be an anhydride, hydrate ananhydride, or hydrate or thereof. solvate thereof. solvate

the the To practice

[01201 To practice

[0120] present present invention invention ROCK inhibitors generallygenerally ROCK inhibitors are without are suitable suitable without so long limitation so limitation as ananinhibitor longas caninhibit inhibitorcan inhibitthe Rho-kinase functionofofRho-kinase thefunction (ROCK), (ROCK), and suitable and suitable

inhibitors 20 inhibitors 20 include include Y-27632 Y-27632 (for example, (for example, refer torefer Ishizaki et al., et to Ishizaki al.,Pharmacol. Mol. 57, 976- 57, Mol. Pharmacol. 976 983 (2000); 983 (2000);Narumiya Narumiya et al., et al., Methods Methods Enzymol. Enzymol. 325,273-284 325,273-284 (2000)), (2000)), sc-3536 sc-3536 (see, e.g.,(see, e.g., Darenfed, H.,etetal. Darenfed,H., al. Cell Cell Motil. Cytoskeleton. Motil.Cytoskeleton. 64:64: 97-109, 97-109, 2007), 2007), Fasudil Fasudil (also(also referred referred to as to as HA 1077) HA1077) (for (for example, example, refer refer to Uenata to Uenata et al., et al., Nature Nature 389: 990-994 (1997)),(1997)), 389: 990-994 H-1152 H-1152 (for (for example,refer example, refertotoSasaki Sasakietetal., al.,Pharmacol. Pharmacol. Ther. Ther. 93: 93: 225-232 225-232 (2002)), (2002)), Wf-536Wf-536 (for example, (for example,

25 to toNakajima refer 25 refer et et Nakajima al., Cancer al., Chemother CancerChemother Pharmacol. Pharmacol. 52(4): 319-324 52(4):319-324 (2003)), Y-30141 (2003)),Y-30141 (describedinin US (described Patent USPatent No.No. 5,478,838) 5,478,838) and derivatives and derivatives thereof, thereof, and antisense nucleicnucleic and antisense acid acid for for ROCK,RNA ROCK, RNA interference interference inducing inducing nucleicacid nucleic acid(for (for example, example, siRNA), siRNA),competitive competitive peptides, peptides, antagonist inhibitoryantibodies, peptides,inhibitory antagonistpeptides, antibody-ScFV antibodies,antibody-ScFV fragments, fragments, dominant negative negative dominant andexpression variants and variants vectors expressionvectors thereof. thereof. Further, Further, since since other other low low molecular molecular compounds compounds are are 30 known 30 known as ROCK as ROCK inhibitors, suchsuch inhibitors, compounds compounds or derivatives or derivatives thereof can can thereof be also be also used in in used the the

present invention present invention(for (forexample, example, refer refer to to United United State State Patent Patent Application Application Nos. 20050209261 Nos. 20050209261, ,

20050192304,20040014755,20040002508,20040002507,20030125344and 20050192304, 20030087919, 20040014755, 20040002508, 20040002507, 20030125344 and 20030087919,

and International and International Patent PatentPublication PublicationNos.2003/062227, Nos.2003/062227,2003/059913, 2003/059913, 2003/062225, 2003/062225,

31

2002/076976 2002/076976 andand In the In 2004/039796). 2004/039796). the present present invention, invention, a combination of one orof a combination two or or one two or moreofofthe more theROCK ROCK inhibitors inhibitors can also can also be be used used

[0121] Additional

[0121] AdditionalROCK ROCK inhibitors inhibitors include, e.g., HA1100, include,e.g., 3-(4-Pyridyl)-IH-indole and HAl100,3-(4-Pyridyl)-1/f-indole and N N-

(4-Pyridyl)-N'-(2,4,6-trichlorophenyl) (4-Pyridyl)-N'-(2,4,6-trichlorophenyl) urea, urea, each each of which of which is commercially is commercially available available (e.g., (e.g., 55 from from Alexis Alexis Biochemicals Biochemicals (Plymouth (Plymouth Meeting, Meeting, PA).PA).

[01221 InInsome some embodiments, ROCK havehave inhibitors the the formula: 2022204080

[0122] embodiments, ROCK inhibitors formula:

N N 1 LLi-L2 2 A A H ZI H B B N N S S R R¹ (I). (I).

In Formula In (I),ring Formula (I), ringAAisisaasubstituted substitutedororunsubstituted cycloalkyl, unsubstitutedcycloalkyl, substituted substituted or unsubstituted or unsubstituted

substitutedororunsubstituted heterocycloalkyl,substituted heterocycloalkyl, unsubstituted aryl, aryl, or or substituted or or substituted unsubstituted unsubstituted heteroaryl. heteroaryl.

10 10 B is B RingRing is a substituted a substituted or unsubstituted or unsubstituted heterocycloalkyl, heterocycloalkyl, or substituted or substituted or unsubstituted or unsubstituted

heteroaryl. heteroaryl.

[0123]

[01231 L¹ is L' -C(O)-NR²- is -C(O)-NR - or -NR2 -C(O)-. or 2-NR²-C(O)-. L 2 is substituted L² is a bond, or unsubstituted a bond, substituted alkylene or unsubstituted alkylene or substituted or or unsubstituted substituted or unsubstitutedheteroalkylene. heteroalkylene.

[0124] R' R² are2 independently hydrogen, substituted or unsubstituted alkyl,

[0124] R¹ and and R are independently hydrogen, substituted or unsubstituted alkyl, 15 substituted 15 substituted or unsubstituted or unsubstituted heteroalkyl, heteroalkyl, substituted substituted or unsubstituted or unsubstituted cycloalkyl, cycloalkyl, substituted substituted or or unsubstitutedheterocycloalkyl, unsubstituted substituted heterocycloalkyl,substituted or unsubstituted or unsubstituted aryl, aryl, or substituted or substituted or or unsubstitutedheteroaryl. unsubstituted heteroaryl.

[01251 In some

[0125] In some embodiments, embodiments, ring ring A is A is a substituted a substituted or unsubstituted or unsubstituted aryl. aryl. Ring Ring A may A may also be also be aa substituted substituted or or unsubstituted unsubstitutedphenyl. phenyl.

20 [0126] 20 [0126] Inembodiments, In other other embodiments, ring ring B is a B is a substituted substituted or unsubstituted or unsubstituted heterocycloalkyl, heterocycloalkyl, or or substituted or substituted or unsubstituted heteroaryl.Ring unsubstitutedheteroaryl. Ring B may B may also also be be a substituted a substituted or unsubstituted or unsubstituted

heteroaryl. InInstill heteroaryl. still other other embodiments, ring embodiments, ring B aissubstituted B is a substituted or unsubstituted or unsubstituted pyrazolyl, pyrazolyl,

substituted or unsubstituted substituted or unsubstitutedfuranyl, substitutedor orunsubstituted furanyl,substituted unsubstituted imidazolyl, imidazolyl, substituted or or substituted unsubstituted substitutedororunsubstituted isoxazolyl,substituted unsubstituted isoxazolyl, unsubstituted oxadiazolyl, oxadiazolyl, substituted substituted or unsubstituted or unsubstituted

25 oxazolyl, 25 oxazolyl, substituted substituted or unsubstituted or unsubstituted pyrrolyl, pyrrolyl, substituted substituted or unsubstituted or unsubstituted pyridyl,pyridyl,

substituted or substituted or unsubstituted unsubstitutedpyrimidyl, pyrimidyl, substituted substituted or or unsubstituted unsubstituted pyridazinyl, pyridazinyl, substituted substituted or or unsubstituted substitutedororunsubstituted thiazolyl,substituted unsubstituted thiazolyl, unsubstituted substitutedor orunsubstituted triazolyl,substituted triazolyl, unsubstituted thienyl, substituted thienyl, or unsubstituted substituted or unsubstituteddihydrothieno-pyrazolyl, dihydrothieno-pyrazolyl, substituted substituted or unsubstituted or unsubstituted

32

thianaphthenyl,substituted thianaphthenyl, substitutedororunsubstituted unsubstituted carbazolyl, carbazolyl, substituted substituted or unsubstituted or unsubstituted

benzothienyl, substitutedororunsubstituted benzothienyl, substituted unsubstituted benzofuranyl, benzofuranyl, substituted substituted or unsubstituted or unsubstituted indolyl, indolyl,

substituted or substituted or unsubstituted unsubstitutedquinolinyl, quinolinyl,substituted substitutedor orunsubstituted unsubstituted benzotriazolyl, benzotriazolyl,

substituted or substituted or unsubstituted unsubstitutedbenzothiazolyl, benzothiazolyl, substituted substituted or unsubstituted or unsubstituted benzooxazolyl, benzooxazolyl,

5 substituted 5 substituted or unsubstituted or unsubstituted benzimidazolyl, benzimidazolyl, substituted substituted or unsubstituted or unsubstituted isoquinolinyl, isoquinolinyl,

substituted or substituted or unsubstituted unsubstitutedisoindolyl, isoindolyl,substituted substitutedororunsubstituted unsubstituted acridinyl, acridinyl, substituted substituted or or 2022204080

benzoisazolyl,or or unsubstitutedbenzoisazolyl, unsubstituted substituted substituted or or unsubstituted unsubstituted dimethylhydantoin. dimethylhydantoin.

2 2

[0127] L²Lmay

[0127] may be be substituted oror unsubstituted substituted unsubstituted CC-C 1 -C 1 o alkyl. In some embodiments, L is alkyl. In some embodiments, L² is

unsubstituted unsubstitutedC-C alkyl. C -C10 alkyl. 2 maybealso L² mayL also substituted or unsubstituted be substituted methylenemethylene or unsubstituted (e.g. (e.g. 1

10 unsubstituted 10 unsubstitutedmethylene). methylene).

1 may C-CC-C

[0128] R²R2may

[0128] may be be hydrogen. hydrogen. R¹ R may be hydrogen be hydrogen or unsubstituted or unsubstituted 10 alkyl. alkyl. In In some some embodiments, R 1isis simply embodiments, R¹ simply hydrogen. hydrogen.

[01291 In some

[0129] In some embodiments embodiments of Formula of Formula (I), ring (I), A isring A is substituted substituted or unsubstituted or unsubstituted aryl, aryl, ring ring BB is is substituted or unsubstituted substituted or unsubstitutedheteroaryl, 1 ishydrogen, heteroaryl,R¹Ris and and hydrogen, L 2unsubstituted L² is is unsubstituted 15 Ci-Cio 15 C-C alkyl. alkyl.

[0130] InInanother

[0130] embodiment,thetheROCK anotherembodiment, ROCK inhibitor inhibitor hashas the formula: theformula:

0 N N- L² L2 NH IZ N ZI (R³) H N_ H N N I 4R' Z (R) (R4r\ yX S S R4 R¹ X (II). y

In Formula In (II), yy isis an Formula(II), an integer integer from andZ zisisananinteger from0 0toto3 3and integerfrom from 0 to 0 to 5. 5. X -N=, X is -CH=-CH= is -N=, or or -CR4=. -CR=. R¹ R 1 and and L2 as L² are aredefined aboveabove as defined in theindefinitions of Formula the definitions (I). of Formula (I).

20 20 [0131]

[0131]R³, R3, R and R are R4 and independently R are independently -CN, -CN, -S(O)R, -S(O)nR6,-NRR, -C(O)R, -NR'R', -NR¹-C(O)R¹, -C(O)R9, -NR -C(O)R", -NR¹²-C(O)-OR¹³, -C(O)NR1 4R -NR -C(O)-ORD,-C(O)NR¹R¹, -NR 15, ¹S(O)R¹, -NR iS(O) R 17, -S(O)NR¹, -OR¹, NR 1 , substituted substituted -OR", -S(O) 2 or 2 or unsubstitutedalkyl, unsubstituted substitutedororunsubstituted alkyl, substituted unsubstituted heteroalkyl, heteroalkyl, substituted substituted or unsubstituted or unsubstituted

substitutedororunsubstituted cycloalkyl, substituted cycloalkyl, unsubstitutedheterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl,aryl, or or substituted or substituted or unsubstituted unsubstitutedheteroaryl, heteroaryl,wherein wherein n isn an is an integer integer fromfrom 0 to 02, towherein 2, wherein if z if Z is is 25 25 greater thanthan greater 1, two 1, R³ R3 moieties twomoieties are optionally joined joined are optionally together to form to together a substituted or form a substituted or cycloalkyl,substituted unsubstitutedcycloalkyl, unsubstituted substitutedor or unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or or

unsubstitutedaryl, unsubstituted aryl, or or substituted substitutedororunsubstituted unsubstitutedheteroaryl. heteroaryl.

33

6 0 11 12 13 1495.1

[0132] R,R,

[0132] R, R', R, R', R', R' R, R¹, , R", R¹¹, R¹²,R, ,R 1R¹, R¹³, R¹, 4 , R", R¹,R, R¹, R", R" R¹ R¹ and andare R19independently are independently hydrogen,substituted hydrogen, substitutedororunsubstituted unsubstituted alkyl, alkyl, substituted substituted or unsubstituted or unsubstituted heteroalkyl, heteroalkyl,

unsubstitutedcycloalkyl, unsubstituted cycloalkyl,substituted substitutedor orunsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or or unsubstitutedaryl, unsubstituted aryl, or or substituted substituted ororunsubstituted unsubstitutedheteroaryl. heteroaryl.

5 [0133] 5 [01331 In some In some embodiments, embodiments, 2 is substituted L² is Lsubstituted or unsubstitutedC-CC alkyl. or unsubstituted -Cio alkyl. L 2 also L² may may also 1

2022204080 be be unsubstituted unsubstitutedC-C C alkyl. Alternatively, -Cio alkyl. 1 Alternatively, L 2 is substituted L² is substituted or unsubstituted methylene or unsubstituted methylene (e.g. unsubstituted (e.g. methylene). unsubstituted methylene).

[01341 InInother

[0134] otherembodiments, embodiments,X X is is-N= -N=oror-CH=. -CH=.TheThe symbol symbol z may Z may be In be 2. 2. still In stillother other embodiments, embodiments, twotwo R 3 moieties R³ moieties at adjacent vertices at adjacent are joined vertices together are joined to from together toafrom substituted or a substituted or 10 10 unsubstitutedheterocycloalkyl. unsubstituted Thesymbol heterocycloalkyl. The symbol z may Z may also also bebe 1.1.The The symbol symbol y may y may be be 0 or 0 or 1. 1.

R3 may R³ may be-OR¹. be-OR".R¹ Rmay be hydrogen 18 may or unsubstituted be hydrogen C-C alkyl. or unsubstituted C1 -Cio alkyl.

[0135]

[01351 In some embodiments, In some L 2 is substituted L² is substituted embodiments, or unsubsituted or unsubsituted methylenemethylene (e.g. substituted (e.g. substituted

methylene),X Xis is-N=-N= methylene), or or -CH=, -CH=, R¹ isR hydrogen, is hydrogen, and y and y are and Z and 0.z are 0.

[01361 InInother

[0136] otherembodiments, embodiments,the thecompounds compoundshashas thethe formula: formula:

0 O N N ZI N IZ N H N HH N

15NN 15 SS .

0 O N IZ ZI N H H N S S

0 O N IZ ZI N NN H H N N o _

NN S (sometimescalledThiazovivinor"Tzv"), (sometimes called Thiazovivin or "Tzv"),

0 O N NNZI H N HN IZ H NNa N H

S S ,or , or

34

O N N N IZ ZI N N H N N

N N S OCH3. OCH.

V. V. GSK3 inhibitors GSK3 inhibitors

2022204080

[01371 InInvarious

[0137] embodiments,oneoneorormore variousembodiments, GSK3 moreGSK3 inhibitors includedininthe inhibitorscancanbebeincluded the mixtures methods,mixtures methods, kitskits andand of of thethe invention. invention. The inventors The inventors have found found have that thethat the inclusion inclusion of of 5 GSK3 5 GSK3 inhibitors inhibitors with at at with least TGFP leastaa TGFß receptor/ALK5 receptor/ALK5 inhibitor,preferably inhibitor, in combination preferably in combination

with a MEK/ERK with pathway MEK/ERK pathway inhibitor inhibitor and in in and particular embodiments, particular RhoGTPase/ROCK embodiments, aaRho GTPase/ROCK inhibitor. Inhibitors of inhibitor. Inhibitors ofGSK3 GSK3 cancan include include antibodies antibodies that that bind,bind, dominant dominant negative negative variantsvariants of, of, siRNA, and siRNA, and microRNA, microRNA, antisense nucleicnucleic antisense acids, acids, and other other polynucleotides andpolynucleotides that target target thatGSK3. GSK3. examplesof of Specific examples Specific GSK3 GSK3 inhibitors inhibitors include, include, butnot but are not limited arelimited to, Kenpaullone, to, Kenpaullone, 1- 1 10 Azakenpaullone, 10 Azakenpaullone, CHIR99021, CHIR99021, CHIR98014, CHIR98014, AR-A014418 AR-A014418 (see, (see, e.g., e.g., et Gould, al., et Gould, al., The The InternationalJournal International Journal ofNeuropsychopharmacology 7:387-390 of Neuropsychopharmacology 7:387-390 (2004)),CTCT (2004)), 99021 99021 (see,e.g., (see, e.g., Wagman,Current Wagman, Current Pharmaceutical Pharmaceutical Design Design 10:1105-1137 10:1105-1137 (2004)), (2004)), CT CT 20026 20026 (see, (see, Wagman, Wagman,

SB216763 supra),SB216763 supra), (see, (see, e.g., e.g., Martin, Martin, et al.,Nature et al., Nature Immunology Immunology 6:777-784 (2005)), (2005)), 6:777-784 AR- AR A014418 (see, A014418 (see, e.g.,Noble, e.g., et et Noble, al., PNAS al.,PNAS 102:6990-6995 lithium lithium (2005)),(2005)), 102:6990-6995 (see,Gould, (see, e.g., Gould, et e.g., et 15 al.,al., 15 Pharmacological Pharmacological Research Research 48:(2003)), 48: 49-53 49-53 SB (2003)), 415286 SB 415286 (see, (see, e.g., e.g., Frame, Frame, et et al., al., Biochemical Biochemical Journal Journal 359:1-16 359:1-16 (2001)) (2001)) and TDZD-8 and TDZD-8 (see, (see, e.g., Chin, et Chin, e.g., et al., Molecular al., Molecular Brain Brain Research, 137(1-2):193-201 Further exemplary (2005)). Further 137(1-2):193-201 (2005)). GSK3inhibitors exemplary GSK3 available from inhibitors available from

Calbiochem Calbiochem (see, (see, e.g.,Dalton, e.g., et et Dalton, al., W02008/094597, al.,WO2008/094597, herein herein incorporated incorporated by reference), by reference),

include but include are not but are notlimited BIO limitedtotoBIO (2'Z,3'£)-6-Bromoindirubin-3'-oxime (2'Z,3')-6-Bromoindirubin-3'-oxime (GSK3 Inhibitor Inhibitor (GSK3 IX); IX); BIO-Acetoxime 20 BIO-Acetoxime 20 (2'Z,3'E)-6-Bromoindirubin-3'-acetoxime (GSK3(GSK3 (2'Z,3'E)-6-Bromoindirubin-3'-acetoxime Inhibitor Inhibitor X); (5-Methyl X); (5-Methyl-

IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor (GSK3-Inhibitor XIII); Pyridocarbazole XIII); Pyridocarbazole-

(GSK3 complex(GSK3 cyclopenadienylruthenium complex cyclopenadienylruthenium Inhibitor XV); InhibitorXV); TDZD-8 TDZD-8 4-Benzyl-2-methyl-,2,4 4-Benzyl-2-methyl-1,2,4-

(GSK3beta thiadiazolidine-3,5-dione(GSK3beta thiadiazolidine-3,5-dione Inhibitor Inhibitor I); 2-Thio(3-iodobenzyl)-5-(-pyridyl)-[,3,4] I); 2-Thio(3-iodobenzyl)-5-(l-pyridyl)-[1,3,4]-

oxadiazole (GSK3beta oxadiazole Inhibitor II); (GSK3beta Inhibitor II);OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione

(GSK3beta 25 (GSK3beta 25 Inhibitor Inhibitor III);alpha-4-Dibromoacetophenone III); alpha-4-Dibromoacetophenone (GSK3beta (GSK3beta Inhibitor Inhibitor VII);VII); AR-AO AR-AO

14418N-(4-Methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea 14418 N-(4-Methoxybenzyl)-N'-(5-nitro-,3-thiazol-2-yl)urea (GSK-3beta (GSK-3beta Inhibitor Inhibitor VIII); 3- VIII); 3 (1-(3-Hydroxypropyl)-IH-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK (1-(3-Hydroxypropyl)-H-pyrrolo[2,3-b]pyridin-3-yl]4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-

InhibitorXI); 3beta Inhibitor XI); TWSI 19 TWSI pyrrolopyrimidine compound 19 pyrrolopyrimidine (GSK3beta compound (GSK3beta Inhibitor L803 XII); L803 InhibitorXII); H-KEAPPAPPQSpP-NH2 H-KEAPPAPPQSpP-NH2 or its or its Myristoylated Myristoylated form (GSK3beta form (GSK3beta Inhibitor XIII);XIII); Inhibitor 2-Chloro-l 2-Chloro-1-

(4,5-dibromo-thiophen-2-yl)-ethanone 30 (4,5-dibromo-thiophen-2-yl)-ethanone 30 (GSK3beta (GSK3beta Inhibitor VI);VI); Inhibitor AR-AO144-18; AR-AO144-18; SB216763; SB216763;

and SB415286. and SB415286. Residues Residues of GSK3b of GSK3b that interact that interact with inhibitors with inhibitors have beenhave been identified. identified. See, See,

35

e.g., Bertrand e.g., et al., Bertrand et Mol Biol. al., J.J.Mol Biol. 333(2): 393-407(2003). 333(2): 393-407 GSK3 (2003).GSK3 inhibitors inhibitors can activate, can activate, for for

Manyofofß-catenin Wnt/B-catenin pathway. Many the Wnt/-catenin example, the example, -catenindownstream genesco-regulate downstreamgenes co-regulate pluripotencygene pluripotency networks. genenetworks. For example, For example, a GSK inhibitor a GSK inhibitor activates activates cMyc expression cMyc expression as as well enhancesitsitsprotein as enhances well as stability and proteinstability activity.Thus, transcriptionalactivity. andtranscriptional in in Thus, some some

5 GSK3GSK3 embodiments, 5 embodiments, inhibitors can can inhibitors be used be used to stimulate to stimulate endogenous MYC MYC endogenous polypeptide polypeptide

expressioninina acell, expression eliminatingthetheneed thereby eliminating cell, thereby forfor need MYCMYC expression expression to induce to induce pluripotency. pluripotency. 2022204080

Those

[01381 Those

[0138] of skill of skill willwill that that appreciate appreciate the the concentration of theof concentration GSK3 GSK3 inhibitor the inhibitor will will dependononwhich depend which specific specific inhibitor inhibitor is used. is used. In certain In certain embodiments, embodiments, a combination of two orof two a combination or differentGSK3 moredifferent more GSK3 inhibitors inhibitors can can be used. be used.

Methods 10 VI.VI.Methods 10 of inducing of inducing pluripotency pluripotency

To date,

[01391 To date,

[0139] a large a large number number of different of different methods methods and protocols have beenhave and protocols been established established

for inducing for non-pluripotent inducing non-pluripotent mammalian mammalian cells into into induced cells induced pluripotent pluripotent stem cells cells (iPSCs). stem(iPSCs). It It is believed is that the believed that agents described the agents hereincancan describedherein be be used used in combination in combination with essentially with essentially any any for generating protocol for protocol generatingiPSCs andand iPSCs thereby thereby improve improve the efficiency of the of the efficiency the protocol. protocol. Thus, Thus, the the 15 present 15 present invention invention provides provides for incubation for incubation of non-pluripotent of non-pluripotent cells cells with with ata TGFß at least least a TGF3 receptor/ALK5inhibitor, receptor/ALK5 witha a combinationwith preferablyinincombination inhibitor, preferably MEK/ERK pathwayinhibitor, MEK/ERK pathway and inhibitor, and in particular in particular Rho GTPase/ROCK embodiments, aaRho embodiments, inhibitorinincombination GTPase/ROCK inhibitor combinationwith anyprotocol withany protocol for generating for generating iPSCs. A selection iPSCs.A selection of protocols of protocols is described is described belowbelow andiseach and each is believed believed to be to be combinable combinable with with thethe agents agents of the of the invention invention to improve to improve efficiency of the of efficiency the protocol. protocol.

[0140] 20 [0140] 20 The improvement The improvement in efficiency of anof in efficiency an iPSC iPSC generation generation protocol willwill protocol depend depend on the on the

andwhich protocol and protocol which agents agents of the of the invention invention are used. are used. In some In some embodiments, embodiments, the efficiency the efficiency is is by at improved by improved least 10%, at least 20%, 50%, 10%, 20%, 75%,100%,150%, 50%, 75%, 200%, 100%, 150%, 200%, 300%300% or more or more compared compared to to the same the sameprotocol without protocolwithout inclusion inclusion of the of the agents agents of the of the invention invention (i.e., TGFßTGF (i.e., receptor/ALK5 receptor/ALK5

inhibitor, MEK/ERK inhibitor, pathway MEK/ERK pathway inhibitor andRho inhibitorand GTPase/ROCK RhoGTPase/ROCK inhibitor). inhibitor). Efficiency Efficiency is is 25 measured 25 withwith measured regard regard to improvement to improvement of the of the number number of iPSCs of iPSCs generated in ainparticular generated time a particulartime frameororthe frame speedbyby thespeed which which iPSCs iPSCs are generated. are generated.

[0141] Studies

[0141] Studies shownshown havehave that retroviral that retroviral transduction transduction of mouse mouse fibroblasts offibroblasts with fourwith four transcription transcription factors that are factors that are highly ESCs expressedin inESCs highly expressed (Oct-3/4, Sox2,Sox2, (Oct-3/4, KLF4 KLF4 and and c-Myc) c-Myc)

generateinduced generate inducedpluripotent pluripotent stem stem (iPS) (iPS) cells. cells. See,See, Takahashi, Takahashi, K. & Yamanaka, K. & Yamanaka, S. Cell S. Cell 126, 126, 30 663-676 30 663-676 (2006); (2006); Okita, Okita, K.,K., Ichisaka,T.T.&&Yamanaka, Ichisaka, Yamanaka,S. S. Nature Nature 448,313-317 448, 313-317 (2007); (2007);

et et Wernig,M.M. Wernig, al.Nature al. Nature 448, 448, 318-324 318-324 (2007); (2007); Maherali, Maherali, N. Cell N. et al. et al.Stem Stem CellCell 55-70 1, 1, Cell 55-70 Meissner,A.,A.,Wernig, (2007); Meissner, (2007); M. &M. Wernig, & Jaenisch, Jaenisch, R. Nature R. Nature Biotechnol. Biotechnol. 25, 1177-1181 25, 1177-1181 (2007); (2007); Takahashi,K.K.etetal. Takahashi, al.Cell 131,861-872 Cell131, 861-872 (2007); (2007); Yu, J. et al. Yu,etJ.al. Science 318, 318, Science 1917-1920 (2007);(2007); 1917-1920

36

Nakagawa, Nakagawa, M. M. et al. et al. Nature Nature Biotechnol. Biotechnol. 26, 101-106 26, 101-106 (2007);(2007); Wernig, Wernig, M., Meissner, M., Meissner, A., A., Cassady,J.P. Cassady, J. P.& & Jaenisch, Jaenisch, R. R. CellCell StemStem Cell. Cell. 2, 10-12 2, 10-12 (2008). (2008). iPS are iPS cells cellssimilar are similar to to ESCs ESCs in in morphology, proliferation,andand morphology, proliferation, pluripotency, pluripotency, judged judged by teratoma by teratoma formation formation and chimaera and chimaera

contribution. contribution.

[0142] 55 [0142] As noted As noted above, above, whilewhile the original the original protocol protocol involved involved introductionofoffour introduction four 2022204080 transcription factors transcription factors into into non-pluripotent non-pluripotentcells, cells,itit has has been beenmore more recently recently discovered discovered that that

sometranscription some transcriptionfactors factorscancan be be omitted. omitted. Thus, Thus, in some in some embodiments, embodiments, the protocols the protocols

involves introducing involves introducingone, one, two two or or three three of of an Oct an Oct polypeptide, polypeptide, a Klfapolypeptide, Klf polypeptide, a Myc a Myc anda aSoxSox polypeptide,and polypeptide, polypeptide polypeptide to non-pluripotent to non-pluripotent underunder cells cells conditions conditions that for that allow allow for 10 thethe 10 non-pluripotentcells non-pluripotent cells to to become iPSCs.For become iPSCs. Forexample, example,each eachofofMaherali andKonrad Maheraliand Konrad Hochedlinger,"Tgfß Hochedlinger, "Tgfp Signal Signal Inhibition Inhibition Cooperates Cooperates in theinInduction the Induction ofand of iPSCs iPSCs and Replaces Replaces Sox2 and Sox2 and cMyc" cMyc"Current Current Biology(2009) Biology andWO/2009/117439 (2009)and WO/2009/117439 describe describe protocols protocols that do do that not require not require all all four four transcription factors to transcription factors to induce inducepluripotency. Moreover, pluripotency.Moreover, the inventors the inventors have have foundthat found that iPSCs iPSCscancan be be generated generated by introducing by introducing Oct4 into Oct4 alone alonecells into and cells and incubating incubating the the 15 cells 15 cellswith witha aTGFß TGFP receptor/ALK5 receptor/ALK5 inhibitor,a aMEK/ERK inhibitor, MEK/ERK pathway pathway inhibitor, inhibitor, a Rhoa Rho GTPase/ROCK GTPase/ROCK inhibitor,and inhibitor, anda ahistone histonedeacetylase deacetylase (HDAC) (HDAC) inhibitor. For inhibitor. Forexample, example, introduction ofofexogenous introduction exogenous Oct4 Oct4 intointo mammalian mammalian cells, cells, in the in the presence presence of a sufficient of a sufficient amount amount of SB431542, of SB431542, PD0325901, PD0325901, Tzv, Tzv, and and valproic valproic acid or acid sodium sodium butyrate, orbutyrate, successfully successfully generated generated iPSCcells. iPSC cells.

[01431 20 [0143] 20 Exemplary Exemplary HDAC inhibitors HDAC inhibitors can include can include antibodies antibodies that bind, that bind, dominant dominant negative negative

variants of, variants of,and siRNA and andsiRNA and antisense antisensenucleic nucleicacids thatthat acids target one or target onemore HDACs. or more HDACs.HDAC HDAC

inhibitors include, but inhibitors include, but are are not not limited to, TSA limited to, TSA(trichostatin (trichostatinA)A) (see, (see, e.g., Adcock, e.g.,Adcock, British British

Journal Journal ofofPharmacology 150:829-831 Pharmacology 150:829-831 (2007)),(2007)), VPA acid) VPA (valproic (valproic e.g.,(see, (see,acid) e.g., et Munster, Munster, et al., Journal al., ofClinical Journal of ClinicalOncology Oncology 25:18S 25:18S (2007): (2007): 1065), 1065), sodiumsodium butyrate butyrate (NaBu) (NaBu) (see, (see, e.g., e.g., 25 Han,Han, 25 et al.,Immunology et al., Immunology Letters108:143-150 Letters 108:143-150 (2007)), SAHA (2007)),SAHA (suberoylanilide (suberoylanilide hydroxamic hydroxamic

acid or acid or vorinostat) vorinostat) (see, (see, e.g., Kelly, et e.g., Kelly, et al., al.,Nature ClinicalPractice Nature Clinical PracticeOncology Oncology 2:150-157 2:150-157

(2005)), sodiumphenylbutyrate (2005)), sodium phenylbutyrate (see, (see, e.g., e.g., Gore, Gore, et al., et al., Cancer Cancer Research Research 66:6361-6369 66:6361-6369

(2006)), depsipeptide(FR901228, (2006)), depsipeptide (FR901228, FK228) FK228) (see, Zhu, (see, e.g., e.g., etZhu, al.,etCurrent al., Current Medicinal Medicinal

Chemistry Chemistry 3(3):187-199 3(3):187-199 (2003)), (2003)), (TPX) (TPX) trapoxin trapoxin (see, Furumai, (see, e.g., e.g., Furumai, et al., et PNAS PNAS 98(1):87 al.,98(1):87- 30 92 92 30 (2001)), (2001)), cyclichydroxamic cyclic hydroxamic acid-containingpeptide acid-containing peptide1 I(CHAP1) (CHAPI) (see,Furumai (see, Furumai supra), supra), MS MS-

(see, e.g., 275 (see, 275 e.g., Carninci, et al., Carninci, et al., W02008/126932, incorporated WO2008/126932, incorporated hereinherein by reference)), by reference)), LBH589 LBH589 (see, e.g., (see, e.g., Goh, et al., Goh, et al.,W02008/108741 incorporated WO2008/108741 incorporated hereinherein by reference) by reference) and (see, and PXD101 PXD101 (see, Goh, supra).In Ingeneral, Goh, supra). general,at atthetheglobal global level,pluripotent level, pluripotent cells cells have have more more histone histone acetylation, acetylation,

and differentiated cells and differentiated cells have haveless lesshistone histoneacetylation. acetylation.Histone Histone acetylation acetylation is also is also involved involved in in

37

regulation of regulation histoneand ofhistone DNA and methylation. In DNA methylation. In some some embodiments, HDAC embodiments,HDAC inhibitors inhibitors

activation of facilitate activation facilitate of silenced silenced pluripotency genes. pluripotency genes.

[0144] To address

[0144] To address the safety the safety issues issues arisearise that that from from target cell cell target genomes genomes harboring harboring integrated integrated

exogenous sequences, exogenoussequences, a number a number of modified geneticgenetic of modified protocols protocols have have been further further developed. beendeveloped. These 5 These 5 protocols protocols produce produce iPSwith iPS cells reduced reduced with potentially cellspotentially risks, risks, and include include non-integrating and non-integrating

2022204080 adenoviruses totodeliver adenoviruses genes genes reprogramming deliverreprogramming (Stadtfeld, al. et M., et M., (Stadtfeld, (2008) ScienceScience al. (2008) 322, 945 322, 945-

949), transient 949), transient transfection transfection ofofreprogramming reprogramming plasmids plasmids (Okita, (Okita, K., etK., al.et(2008) al. (2008) Science Science 322, 322, 949-953),piggyBac 949-953), piggyBac transposition transposition systems systems (Woltjen, (Woltjen, K., et K., al. et al. (2009). (2009). NatureNature 458, 766-770, 458, 766-770,

Kaji, K., Kaji, et al. K., et (2009) Nature al. (2009) 458,771-775), Nature 458, 771-775), Cre-excisable Cre-excisable viruses viruses (Soldner, F., etF., (Soldner, al.et (2009) al. (2009) 10 CellCell 10 136,136,964-977), oriP/EBNA l -based andoriP/EBNA1-based 964-977),and episomal episomal expression expression system J., J., (Yu,(Yu, system et et al. (2009) al. (2009) ScienceDOI: Science DOI: 10.1126). 10.1126). Furthermore, Furthermore, strategies strategies of exploiting of exploiting endogenous endogenous gene expression gene expression in in cell types certain cell certain also allowed types also allowedeasier reprogramming easierreprogramming and/or and/or with less less required withrequired exogenous exogenous

genes (Shi, Y., genes(Shi, et al. Y., et al. (2008b). CellStem (2008b). Cell Cell StemCell 2,2,525-528; 525-528; Aasen, Aasen, T.,al. T., et et al. (2008) (2008) Nat Nat 26,1276-1284; Biotechnol26, Biotechnol 1276-1284; J.B.,J.B., Kim,Kim, et (2008). et al. al. (2008). Nature Nature 454, 646-650). 454, 646-650). Moreover, Moreover, small small 15 molecules molecules have have been been identified thatenhance identifiedthat reprogramming enhancereprogramming efficiency andreplace efficiencyand certain replacecertain reprogramming reprogramming factors factors Y., Y., (Shi, (Shi, et al. et al. (2008) (2008) StemStem CellCell Cell Cell 2, 525-528, 2, 525-528, Shi,etY., Shi, Y., al.. al.. (2008) et (2008) Cell et et 568-574,Li,Li,W.,W., Cell3,3,568-574, StemCell Cell Stem al.al. (2009) (2009) Cell Cell Stem Stem CellCell 4, 16-19; 4, 16-19; Huangfu, D., et D., Huangfu, al. et al. (2008) NatBiotechnol (2008) Nat Biotechnol26,26, 1269-1275, 1269-1275, Huangfu, D., et D., Huangfu, al. et al. (2008) (2008) Nat Biotechnol 26, 795- 26, Nat Biotechnol 795 797). 797).

20 [0145] 20 [0145] recently, recently, Moreover, Moreover, it has it has been been shown thatshown transcription factors can factors that transcription can beasdelivered be delivered as exogenous exogenous protein protein to to non-pluripotent non-pluripotent cells, cells, to generate to generate iPSCs. iPSCs. See, e.g., See, e.g., WO/2009/117439; WO/2009/117439;

Zhouetetal., Zhou al., Cell StemCell Cell Stem 4:381-384 Cell4:381-384 (2009). (2009). One can can introduce Oneintroduce an exogenous an exogenous polypeptide polypeptide

(i.e., a aprotein (i.e., proteinprovided fromoutside provided from thecell outsidethe and/orthat celland/or notproduced thatisisnot producedby by the the cell) cell) into into thethe

cell by cell a number by a number ofof differentmethods different thatthat methods do not do not involve involve introduction introduction of a polynucleotide of a polynucleotide

25 encoding 25 the the encoding polypeptide. in in Thus, polypeptide.Thus, some some embodiments, embodiments, non-pluripotent non-pluripotent cellsarearecontacted cells contacted with aa TGF$ with TGFß receptor/ALK5 preferably in inhibitor, preferably receptor/ALK5inhibitor, combination with in combination with aaMEK/ERK pathway MEK/ERK pathway

inhibitor, and inhibitor, andin in particular embodiments, particular a Rho embodiments, GTPase/ROCK a Rho GTPase/ROCK inhibitor and one inhibitor and or more one or more

exogenoustranscription exogenous transcription factor factor proteins, proteins, e.g.,one, e.g., one, two, two, three three or or allall four four of of an an OctOct polypeptide, polypeptide,

a Klf a polypeptide,a aMyc Klf polypeptide, Myc polypeptide, polypeptide, a Soxa polypeptide. and and Sox polypeptide.

[01461 30 [0146] 30 A variety A variety have have of ways of ways been been described described for introducing for introducing the the relevantprotein relevant factors proteinfactors into into the cells. In target cells. the target In one embodiment, one embodiment, introduction of aof introduction polypeptide into ainto a polypeptide cell can cella can

comprise of of introduction compriseintroduction a polynucleotide a polynucleotide comprising moreorexpression one or one comprising cassettescassettes more expression into a into a cell and cell inducingexpression, and inducing thereby expression,thereby introducing introducing the polypeptides the polypeptides into the the bycell intocell by andtranslation transcription and transcription translationfrom expression fromthetheexpression cassette. cassette.

38

[0147] Alternatively, one or more proteins can simply be cultured in the presence of target

[01471 Alternatively, one or more proteins can simply be cultured in the presence of target cells under cells conditionstotoallow under conditions of of introduction allowforforintroduction thethe proteins proteins into thethe into cell. See, cell.See, e.g.,Zhou e.g., H H Zhou et al.,Generation et al., Generation of ofinduced stem pluripotent stem inducedpluripotent cells cells using using recombinant recombinant proteins. proteins. Cell Stem Cell Stem

2009 May Cell. 2009 Cell. May 8;4(5):381-4. In some 8;4(5):381-4. In some embodiments, exogenousproteins the exogenous embodiments,the the comprise the proteins comprise transcription 55 transcription factor factor polypeptide polypeptide of interest of interest linked (e.g.,(e.g., linked linked as a as linked a fusion fusion protein protein or otherwise or otherwise

covalently non-covalently covalentlyorornon-covalently linked) linked) topolypeptide to a that that a polypeptide enhances enhances the ability the ability of the of the 2022204080

transcription to enter factor to transcription factor enter the cell (and the cell in some (and in embodiments someembodiments the cell the cell nucleus). nucleus).

[0148] Examples

[0148] of of Examples sequences polypeptidesequences polypeptide that enhancetransport thatenhance membranes across membranes transport across include, but are include, but not limited are not to, the limited to, Drosophila the Drosophila homeoprotein homeoprotein antennapedia antennapedia transcription transcription

10 protein 10 protein (AntHD) (Joliot(Joliot (AntHD) etNew et al., al.,Biol. Biol. New 3: 1121-34,1991 ; Joliot ;et 3: 1121-34,1991 al., et Joliot Proc. Acad. Natl.Sci. Proc.Acad. al., Natl. Sci. USA, 88:1864-8,1991 USA, 88: 1864-8,1991 Le Roux al., et ; LeetRoux al., Proc. Proc. Natl. Acad. Acad. Natl. Sci. Sci.90:USA, USA, 90: 9120-4,1993), 9120-4,1993), the the herpessimplex herpes virusstructural simplexvirus protein structuralprotein VP22 VP22 (Elliott and and (Elliott O'Hare, O'Hare, Cell 88: 88: 223-33,1997); Cell223-33,1997); the the activatorTATTAT transcriptionalactivator HIV-1transcriptional HIV-1 protein protein (Green (Green and Loewenstein, Cell 55: Cell and Loewenstein, 55: 1179-1188, 1179-1188,

1988Frankel 1988 and and ; Frankel Pabo, 55: 155: Cell Cell Pabo, 289-1193, deliverydelivery 1988); 1988); 1 289-1193, enhancing enhancing transporters transporters such as such as 15 described 15 US Patent in USinPatent described No. 6,730,293 No. 6,730,293 (including (including but nottolimited but not limited to ansequence an peptide peptide sequence comprisingatatleast comprising least7-25 contiguous 7-25contiguous arginines); and and arginines); commercially commercially available available PenetratinTM Penetratin 1 I peptide, andthe peptide, and Peptide DiatosPeptide theDiatos Vectors Vectors ("DPVs") of theof ("DPVs") Vectocell® availableavailable platformplatform the Vectocell@ from from Daitos S.A. Daitos of Paris, S.A. of Paris, France.See France. also, See WO/2005/084158 also, WO/2005/084158 and WO/2007/123667 and WO/2007/123667 andand

transportersdescribed additional transporters additional onlyonly therein.NotNot describedtherein. can these can these proteins proteins pass through pass through the the 20 plasma 20 plasma membrane membrane but the attachment but the attachment of other proteins, as thesuch proteins, of other such as the transcription transcription factors factors described herein,isis sufficient described herein, stimulatethe to stimulate sufficient to uptakeofofthese cellular uptake thecellular complexes. thesecomplexes.

In some

[01491 In some

[0149] embodiments, embodiments, the transcription the transcription factor polypeptides factor polypeptides herein are herein described described are exogenously exogenously introduced introduced as part of aof as part a liposome, liposome, or lipid or lipid cocktail such such cocktail as commercially as commercially

Fugene6 available Fugene6 available andand Lipofectamine). Lipofectamine). In another In another alternative, alternative, the transcription the transcription factor factor 25 proteins 25 proteins be microinjected canmicroinjected can be or otherwise directlydirectly or otherwise introduced into the into introduced target target thecell. cell.

[01501 AsAs

[0150] discussed the Examples discussedininthe WO/2009/117439, ExamplesofofWO/2009/117439, incubation incubation of cells the withthe of cellswith transcription polypeptidesofof factor polypeptides transcription factor the forfor invention theinvention extended extended periods periods is toxic is toxic to the to the cells. cells.

Therefore, thepresent Therefore,the invention presentinvention provides for for provides intermittent intermittent incubation incubation of non-pluripotent of non-pluripotent

mammalian mammalian cells cells with one one with or more or more of apolypeptide, of a Klf an Oct an Klf polypeptide, Oct polypeptide, polypeptide, a Myc a Myc 30 30 polypeptide, and/or polypeptide, and/or Sox polypeptide, a Sox apolypeptide, with intervening with intervening periods periods of of incubation incubation ofin of the cells the cells in the oneorormore theone absenceofofthe the absence more polypeptides. polypeptides. In embodiments, In some the cycle the some embodiments, cycle of incubation of incubation

with and with can can polypeptides withoutthethepolypeptides andwithout be repeated be repeated for 2, or more for3,2,4,3,5,4,6,5, or6, more and isand timestimes is performedforforsufficient performed of of lengths sufficientlengths time time (i.e.,the (i.e., incubations theincubations with and and with without without proteins) proteins) to to the development achieve the achieve development of pluripotentcells. of pluripotent cells.Various Variousagents (e.g., agents (e.g., MEK/ERK pathway MEK/ERK pathway

39

and/orGSK3 inhibitor and/or inhibitor inhibitorand/or GSK3 inhibitor and/orTGFbeta/ALK5 inhibitor and/or TGFbeta/ALK5 inhibitor Rho GTPase/ROCK and/or Rho GTPase/ROCK

inhibitor)cancanbe be pathwayinhibitor) pathway included included to improve to improve efficiency of theofmethod. efficiency the method.

[0151] TheThe

[0151] variousinhibitors various (e.g., TGFP inhibitors (e.g., TGFß receptor/ALK5 inhibitor, MEK/ERK receptor/ALK5 inhibitor, pathway MEK/ERK pathway

inhibitor,and inhibitor, andin in particular embodiments, particular RhoRhoGTPase/ROCK embodiments, inhibitor, and/or GTPase/ROCK inhibitor, and/orGSK3 GSK3

inhibitor, 5 inhibitor, 5 cancan etc.) etc.) be be contacted contacted to non-pluripotent cells cells to non-pluripotent either either priorprior to, simultaneous to, simultaneous with, with, or or 2022204080 after after delivery of, programming delivery of, transcription programming transcription factors (for(for factors example, example, delivered delivered via expression via expression

cassette cassette or as proteins). or as Forconvenience, proteins). For convenience,thethe dayday the the reprogramming factorsfactors reprogramming are delivered are delivered is is designated "day1".1".In In designated "day some some embodiments, embodiments, the inhibitors the inhibitors are contacted to cellstoincells are contacted in aggregate aggregate

(i.e., asasa a"cocktail") (i.e., days 3-7 about days "cocktail")atatabout 3-7 and continuedforfor7-14 and continued 7-14 days. days. Alternatively, Alternatively, in some in some

10 embodiments, 10 embodiments, the cocktail the cocktail is contacted is contacted to theatcells to the cells day at day 0 (i.e., 0 (i.e., day before a day abefore the the preprogramming factors) preprogramming factors) and and incubated incubated for about for about 14-30 days. 14-30 days.

In other

[0152] In other

[0152] embodiments, embodiments, different different inhibitors inhibitors are at are added added at different different times. times. In some In some embodiments, embodiments, at at 1-71-7 days days after after the the delivery delivery of the of the reprogramming reprogramming factors, factors, the are the cells cells are contacted with contacted with compound combinationofofananTGFß compound combination TGFreceptor/ALK5 receptor/ALK5 inhibitor inhibitor (e.g., (e.g.,

15 SB431542) SB431542) and and a a ROCK ROCK inhibitor inhibitor for days, for 1-8 1-8 days, followed followed by contacting thethe by contacting cellswith cells the withthe TGF$receptor/ALK5 TGFß receptor/ALK5 inhibitor,ROCK inhibitor, ROCK inhibitorand inhibitor and a a MEK/ERK MEK/ERK pathway pathway inhibit inhibit (e.g., (e.g.,

PD0325901) PD0325901) for for 1-8 1-8 This This days. days. can can be be optionally optionally followed followed by contact by contact with the with TGFß the TGF3 inhibitor and receptor/ALK5inhibitor receptor/ALK5 and MEK/ERK pathway MEK/ERK pathway inhibitor inhibitor (but (but notnecessarily not the ROCK necessarily the ROCK inhibitor) for inhibitor) 1-4 days, for 1-4 days, followed contact followedbybycontact with thethe with MEK/ERK MEK/ERK inhibitor inhibitor pathway pathway (but not (but not 20 thethe 20 TGF TGFß receptor/ALK5 receptor/ALK5 inhibitor inhibitor or ROCK or ROCK inhibitor), and and inhibitor), optionally optionally finally withbasal finallywith basal (e.g., basal (e.g., human) ESES basal human) medium medium without without inhibitors inhibitors fordays. for 1-4 1-4 days. Other combinations Other combinations can also can also be employed. be employed.

IV. IV. Transformation Transformation

[01531 ThisThis

[0153] invention invention employs routineroutine employs techniques techniques in theoffield in the field of recombinant recombinant genetics. genetics. 25 BasicBasic 25 textstexts disclosing disclosing the general the general methods methods of use in use in ofthis invention include include this invention Sambrook Sambrook et al., et al., Molecular Cloning, AA Laboratory MolecularCloning, Manual LaboratoryManual (3rded. (3rd ed. 2001); Kriegler, Gene 2001); Kriegler, Transfer and Gene Transfer and

Expression: AA Laboratory Expression: Manual LaboratoryManual (1990); andCurrent (1990);and Current Protocols Protocols Molecular ininMolecular Biology Biology

(Ausubel (Ausubel etetal., al., eds., eds., 1994)). 1994)).

In some

[01541 In some

[0154] embodiments, embodiments, the species of cell of the species andcell and to protein protein to be expressed be expressed is the is the same. same. 30 For For 30 example, example, if a mouse if a mouse cell iscell is used, used, mouse ortholog a mousea ortholog is introduced is introduced into the into the cell. If acell. humanIf a human is used, cell is cell used, aa human orthologis isintroduced human ortholog introduced into into thethe cell. cell.

40

[0155] It will

[0155] It will be be appreciated appreciated thatthat where where two two or or proteins more more proteins are to are to be expressed be expressed in in a cell, a cell, one or one expression multipleexpression or multiple cassettes be be cancan cassettes used. used. For example, where where For example, one expression one expression cassette cassette polypeptides, multiplepolypeptides, expressesmultiple expresses a polycistronic a polycistronic expression expression cassette cassette can be be used. canused.

A. A. Plasmid Vectors Plasmid Vectors

55 [0156] In certain

[01561 embodiments, embodiments, In certain a plasmid a plasmid vector vector is contemplated forfor is contemplated use transform aa usetototransform 2022204080

host In general, cell. In host cell. plasmidvectors general, plasmid containing vectorscontaining replicon and and replicon control control sequences sequences which which are are derived fromspecies derived from withwith compatible speciescompatible the host cell cell the host are used are used in connection in connection with these these with hosts. hosts. The carrya areplication cancarry vectorcan Thevector site,asaswell replicationsite, marking wellasasmarking sequences whichwhich sequences are capable are capable of of phenotypic providingphenotypic providing selection selection in transformed in transformed cells. cells.

10 10 B. B. Viral Vectors Viral Vectors

[0157] The The

[0157] ability ability of certain of certain viruses viruses to infect to infect cells cells or enter or enter cells cells viavia receptor-mediated receptor-mediated

endocytosis, andtotointegrate endocytosis, and integrateinto hostcell intohost genome cellgenome and and express genesgenes viralviral express and and stablystably

efficiently have efficiently madethem have made them attractive attractive candidates candidates for the for the transfer transfer of foreign of foreign nucleic acidsacids nucleic into into cells (e.g., cells (e.g.,mammalian Non-limiting cells).Non-limiting mammalian cells). examples examples of virus of virus vectors vectors that may used be thatbemay to used to deliver 15 deliver 15 a nucleic acid acid a nucleic of the the present of present invention invention are described below. below. are described

i. Adenoviral i. Vectors AdenoviralVectors

[0158] A particular

[0158] A particular method method for delivery of theof for delivery the nucleic nucleic acid involves the use the acid involves use of an of an expression adenovirusexpression adenovirus vector. vector. Although Although adenovirus vectorsvectors adenovirus aretoknown are known lowhave have a to a low capacity capacity for integration for integration into DNA, genomicDNA, into genomic this this feature feature is counterbalanced by theby is counterbalanced high high efficiency theefficiency of of 20 genegene 20 transfer transfer afforded afforded by these by these vectors. vectors. "Adenovirus expressionexpression "Adenovirus meant to isinclude vector" is vector" meant to include those constructs those adenovirus containingadenovirus constructscontaining sequences sequences sufficient (a) support to (a)tosupport sufficient packaging packaging of the of the construct and(b) construct and ultimatelyexpress (b)totoultimately a tissueororcell-specific expressa tissue construct cell-specificconstruct that that has has been been cloned cloned

Knowledge therein. Knowledge therein. of the of the genetic genetic organization organization or adenovirus, or adenovirus, a -36 a ~36 kb, linear, double-double kb, linear, DNA strandedDNA stranded virus, virus, allows allows substitution substitution of large of large pieces pieces of adenoviral of adenoviral DNA DNA with with foreign foreign 25 sequences 25 sequences 7 kb up 7to kb up to et et (Grunhaus (Grunhaus al., Seminarinin Virology, al.,Seminar 1992)). 200(2):535-546, 1992)). Virology, 200(2):535-546,

ii. Vectors AAVVectors ii. AAV

[0159] The nucleic acid acid

[01591 The nucleic may be be introduced introduced may cellthe into theinto using using adenovirus celladenovirus assisted assisted transfection. transfection. Increased transfectionefficiencies Increasedtransfection have efficiencieshave been been reported reported in cell in cell systems usingusing systems coupled adenoviruscoupled adenovirus systems systems (Kelleher (Kelleher and Vos, Vos, Biotechniques, and Biotechniques, 17(6):1110-7, 17(6):1110-7, 1994; 1994; Cotten et Cotten et 30 30 al.,Proc al., Proc NatlAcad Natl Acad SciUSA, Sci USA,89(13):6094-6098, 89(13):6094-6098,1992; 1992; Curiel,Nat Curiel, NatImmun, Immun, 13(2-3):141-64, 13(2-3):141-64,

Adeno-associated 1994.). Adeno-associated 1994.). virus virus (AAV) (AAV) is an attractive is an attractive system system vectorvector as ait high as it has has a high it itcan integrationandand frequencyofofintegration frequency caninfect non-dividing infectnon-dividing cells, cells, thus thus making making it useful it useful for for

41

delivery genesinto ofgenes delivery of mammalian intomammalian cells, cells, for for example, example, in tissue in tissue culture culture (Muzyczka, Curr TopCurr Top (Muzyczka, Microbiol Microbiol Immunol, Immunol, 158:97-129, 1992) 1992) 158:97-129, or in vivo. vivo. Details or in Details concerning concerning the generation the generation and use and use of rAAV of rAAV areare vectors vectors described described in U.S. Pat. Pat. in U.S. Nos.Nos. 5,139,941 5,139,941 and 4,797,368, and 4,797,368, each incorporated each incorporated

byreference. herein by herein reference.

5 5 iii. Retroviral iii. Vectors Retroviral Vectors 2022204080

[0160] Retroviruses

[0160] have have Retroviruses promise promise as geneasdelivery vectors vectors gene delivery due to due to their their to ability ability to integrate integrate

their their genes into the genes into the host host genome, genome, transferring transferring a large a large amount amount of foreign of foreign genetic genetic material, material,

infecting aa broad infecting spectrum broadspectrum of of species cellcell andand species types and and types of being of being packaged packaged in special in special cell- cell lines (Miller lines et al., (Miller et Am. J.J. Clin. al.,Am. Clin. Oncol., Oncol., 15(3):216-221, 1992). 15(3):216-221,1992).

10 [0161] 10 [0161] In toorder In order to construct construct a retroviral vector,vector, a retroviral a nucleic a nucleic acid (e.g., (e.g., acid one encoding gene of gene of one encoding interest) isis inserted interest) intothe inserted into viralgenome the viral in the genome in placeofofcertain the place certainviral sequencesto toproduce viral sequences produce a a that is virus that virus is replication-defective. replication-defective. To produce Toproduce virions, virions, a packaging cell cell a packaging lineline containing containing the the pol, and gag, pol, gag, envgenes and env genesbutbut without without LTRLTR the the and packaging and packaging components components is constructed is constructed

(Mannetet al., (Mann al., Cell, Cell,33:153-159, 33:153-159,1983). 1983).When When aa recombinant recombinant plasmid plasmid containing containing aacDNA, cDNA,

15 together 15 with with together the retroviral the retroviral LTRpackaging LTR and sequencessequences and packaging is introduced is introduced into cell into a special a special cell line (e.g., line (e.g.,by by calcium calcium phosphate precipitationforfor phosphateprecipitation example), the the example), packaging packaging sequence sequence allows allows the RNA the RNA of of transcript transcript thethe recombinant recombinant plasmid to be to plasmid be packaged packaged into viral viral particles, intoparticles, which which are are intothe secretedinto then secreted then theculture media culturemedia (Nicolas and and (Nicolas Rubinstein, Rubinstein, In: Vectors: In: Vectors: A survey A survey of of molecular molecular cloning cloning vectors vectors andand their their uses, uses, Rodriguez Rodriguez and Denhardt, and Denhardt, eds., Stoneham: eds., Stoneham:

20 Butterworth, 20 pp.pp. Butterworth, 494-513, 494-513, 1988; 1988; Temin, Temin, In:In: Gene Gene Transfer, Transfer, Kucherlapati (ed.), New Kucherlapati(ed.), York: New York:

149-188, Press,pp.pp.149-188, PlenumPress, Plenum Mann Mann 1986; 1986; al., Cell, et al.,etCell, 33:153-159, 33:153-159, 1983). 1983). The The media media therecombinant containingthe containing recombinant retroviruses retroviruses is then is then collected, collected, optionally optionally concentrated, concentrated, and and used used for gene for gene transfer. transfer. Retroviral Retroviralvectors vectorsareareable able to to infecta broad infect a broad variety variety of of celltypes. cell types. However, However,

integration and integration andstable stableexpression expressiontypically typically involves involves the the division division of host of host cells cells (Paskind (Paskind et al., et al.,

25 Virology, 25 Virology, 67:242-248, 67:242-248, 1975). 1975).

[01621 Lentiviruses

[0162] Lentiviruses are complex are complex retroviruses, which, which, retroviruses, in addition in addition to the common common retroviral to the retroviral gag,pol, genesgag, genes pol,and env,contain andenv, contain other other genes withwith genes regulatory regulatory or structural or structural function. function. Lentiviral Lentiviral

are well vectors are vectors known well known in the artart in the (see, example, (see,forforexample, Naldini Naldini et al., et al., Science, Science, 272(5259):263 272(5259):263-

267, 1996; 267, 1996;Zufferey Zuffereyet et al.,Nat al., NatBiotechnol, Biotechnol, 15(9):871-875, 15(9):871-875, 1997; 1997; BlomerBlomer et al., et al., J Virol., J Virol.,

30 30 71(9):6641-6649, 71(9):6641-6649, 1997; 1997; U.S. U.S. Patent Patent Nos. Nos. 6,013,516 andand 6,013,516 5,994,136). Some 5,994,136).Some examples examples of of lentivirus include lentivirus Human thethe include Human Immunodeficiency Immunodeficiency Viruses: HIV-2andandthe HIV-1,HIV-2 Viruses: HIV-1, theSimian Simian Immunodeficiency Immunodeficiency Virus: Virus: SIV. SIV. Lentiviral vectorsvectors Lentiviral have have been been generated generated by by multiply multiply

42

HIVvirulence the HIV attenuating the attenuating virulence genes, genes, for for example, example, the genes the genes env, vif, vif, vpr, env, vpr, vpunef vpu and nef are andare making deleted making deleted thethe vector vector biologically biologically safe. safe.

[0163] Recombinant lentiviral vectors

[01631 Recombinant lentiviral are capable are capable vectors of infecting of infecting non-dividing cells andcells non-dividing can and can be bothininvivo forboth usedfor be used andexexvivo vivoand gene vivogene transfer andand transfer expression expression of nucleic acid acid of nucleic sequences. sequences.

5 For For 5 example, example, recombinant recombinant lentivirus capable capable lentivirus of infecting of infecting a non-dividing a non-dividing cella wherein cell wherein a

2022204080 suitable host suitable host cell cell is is transfeceted transfected with twoorormore with two more vectors vectors carrying carrying the the packaging packaging functions, functions,

namely gag, namely gag, pol pol andand env, env, as well as well as rev as rev and and tat tat is described is described in U.S. in U.S. Patent Patent No. 5,994,136, No. 5,994,136,

incorporatedherein incorporated One One reference. hereinbybyreference. may target may target the recombinant virus byvirus the recombinant linkage linkage by of the of the envelopeprotein envelope withan an proteinwith antibody or aor antibody a particular particular ligand for for ligand targeting to ato targeting receptor of a of a a receptor particular 10 particular 10 cell-type. cell-type. By inserting By inserting a sequence a sequence (including (including a regulatory region) region) a regulatory of interest of interest into into the viral the vector, along viral vector, along with gene anothergene withanother which which encodes encodes the ligand for a for the ligand a receptor receptor on a specific on a specific

cell, for target cell, target for example, example, the vectorisis now the vector target-specific. nowtarget-specific.

iv. Delivery iv. DeliveryUsing Modified UsingModified Viruses Viruses

[01641 A nucleic

[0164] acid acid A nucleic be delivered to betodelivered may may be be housed housed within within an an infective infective virus that has that virus been has been engineered 15 engineered 15 to express to express a specific a specific binding ligand.ligand. binding The The virus virus particle particle will will thus thus bind bind specifically specifically

the cognate to the to receptorsofofthe cognate receptors targetcell thetarget deliverthethecontents anddeliver cell and to to contents the cell.A A thecell. novel novel

approach to to designed approachdesigned allow allow specific specific targeting targeting of retrovirus of retrovirus vectors vectors was developed was developed based based on on the chemical the modification chemicalmodification of a a retrovirus ofretrovirus by the by the chemical chemical addition addition of lactose of lactose residues to theto residues the viral envelope. viral envelope. This modification Thismodification cancan permit permit the specific the specific infection infection of hepatocytes of hepatocytes via via 20 sialoglycoprotein 20 receptors. sialoglycoproteinreceptors.

Another

[0165] Another

[0165] approach approach to targeting to targeting of recombinant of recombinant retroviruses retroviruses was indesigned was designed which in which biotinylated againsta aretroviral antibodiesagainst biotinylated antibodies envelope retroviralenvelope protein and and protein against against a specific cell cell a specific receptor used.TheThe wereused. receptor were antibodies were were antibodies coupled via thevia coupled the biotin biotin components components by using by using streptavidin (Roux streptavidin al.,Proc. (Rouxetetal., Proc.Nat'l Acad.Sci. Nat'lAcad. USA, Sci.USA, 86:9079-9083, 1989). 1989). 86:9079-9083, Using antibodies Using antibodies

25 against 25 against major major histocompatibility complexcomplex histocompatibility class class I and class II class I and II antigens, antigens, they demonstrated they demonstrated the the of aa variety infection of infection of human variety of humancells those borethose thatbore cellsthat surface surface antigens withwith antigens an ecotropic virusvirus an ecotropic in vitro in vitro (Roux etal., (Roux et al., 1989). 1989).

C. C. Vector Vector Delivery andCell Delivery and Transformation Cell Transformation

Suitable

[0166] Suitable

[0166] methods methods for nucleic for nucleic acid delivery acid delivery for transformation for transformation of aa cell, of a cell, tissuea tissue or an or an organism 30 organism 30 use the forwith for use the current withcurrent invention invention are believed are believed to include include virtually to virtually anybymethod any method by whicha anucleic which acid(e.g., nucleicacid DNA) (e.g.,DNA) can can be introduced into ainto be introduced a cell, cell, a tissue an organism, or anororganism, a tissue as as hereinororasaswould describedherein described wouldbe be known known one to onetoof of ordinary ordinary skill in theinart. skill the Such Such methods art. methods

43

include, but include, are not but are not limited limited to, direct delivery to, direct delivery of ofDNA DNA such such as ex as by ex vivo by vivo transfection transfection (Wilson (Wilson

et al., et al.,Science, Science, 244:1344-1346, 1989, 244:1344-1346, 1989, Nabel Nabel and and Baltimore, Baltimore, NatureNature 326:711-713, 326:711-713, 1987), 1987), optionally with optionally withFugene6 Fugene6 (Roche) (Roche) or Lipofectamine or Lipofectamine (Invitrogen), (Invitrogen), by injection by injection (U.S. (U.S. Patent Patent Nos. 5,981,274, 5,994,624,5,981,274, Nos. 5,994,624, 5,945,100, 5,945,100, 5,780,448, 5,780,448, 5,736,524, 5,736,524, 5,702,932, 5,702,932, 5,656,610, 5,656,610, 5,589,4665,589,466

55 and and 5,580,859, 5,580,859, each incorporated each incorporated herein herein by reference), includingincluding by reference), microinjection microinjection (Harland (Harland and and CellBiol., Weintraub,J.J.Cell Weintraub, Biol.,101:1094-1099, 101:1094-1099, 1985; 1985; U.S. U.S. Pat.5,789,215, Pat. No. No. 5,789,215, incorporated incorporated herein herein 2022204080

by reference); bybyelectroporation by reference); (U.S.Pat. electroporation(U.S. Pat. No.No. 5,384,253, 5,384,253, incorporated hereinherein incorporated by reference; by reference;

Tur-Kaspa Tur-Kaspa et et al.,Mol. al., Mol.Cell Cell Biol.,6:716-718, Biol., 6:716-718, 1986; 1986; Potter Potter et al., et al., Proc. Proc. Nat'l Nat'l Acad. Acad. Sci.Sci. USA,USA,

81:7161-7165, 1984); 81:7161-7165, 1984); by by calcium calcium phosphate phosphate precipitation precipitation (Graham (Graham and Van Der Eb, Van Der Eb, 10 Virology, 10 Virology, 52:456-467, 52:456-467, 1973; 1973; Chen Chen andand Okayama, Mol.Mol. Okayama, CellCell Biol., Biol., 7(8):2745-2752, 1987; 7(8):2745-2752,1987; Rippeetetal., Rippe al., Mol. CellBiol., Mol. Cell Biol., 10:689-695, 1990); 10:689-695,1990); by by using using DEAE-dextran DEAE-dextran followedfollowed by by polyethyleneglycol polyethylene (Gopal, glycol(Gopal, Mol. Mol. CellCell Biol., Biol., 5:1188-1190, 1985);1985); 5:1188-1190, by direct by direct sonic loading sonic loading

(Fechheimeret et (Fechheimer al.,Proc. al., Proc.Nat'l Nat'lAcad. Sci.USA, Acad. Sci. USA, 84:8463-8467, 84:8463-8467, 1987); 1987); by liposome by liposome mediated mediated transfection (Nicolau transfection (Nicolauandand Sene, Sene, Biochim. Biochim. Biophys. Biophys. Acta, Acta, 721:185-190, 721:185-190, 1982;etFraley 1982; Fraley al., et al., 15 Proc. 15 Proc. Nat'lAcad. Nat'l Sci. USA, Acad. Sci. USA,76:3348-3352, 76:3348-3352,1979; Nicolauetetal., 1979;Nicolau al., Method5 Methods Enzymol., 149:157 Enzymol., 149:157- 176, 1987; 176, Wong 1987;Wong et al., et al., Gene, Gene, 10:87-94, 10:87-94, 1980;1980; Kaneda Kaneda et al., et al., Science, Science, 243:375-378, 243:375-378, 1989; 1989; Katoetetal., Kato al., JBiol. J Biol. Chem., 266:3361-3364, Chem., 266:3361-3364, 1991) 1991) and receptor-mediated and receptor-mediated transfection transfection (Wu and (Wu and Wu, Biochemistry, Wu, 1988; Wu 27:887-892, 1988; Biochemistry, 27:887-892, Wuand Wu,J.J.Biol. andWu, Biol. Chem., Chem., 262:4429-4432, 1987); 262:4429-4432,1987); and any and anycombination combination of such of such methods, methods, each each of of is which which is incorporated incorporated herein herein by by reference. reference.

20 20

VII. Mixtures VII. Mixtures

[0167] TheThe

[0167] present present invention invention provides provides for mixtures for mixtures that improve that improve the efficiency the efficiency of of generationofofiPSCs generation iPSCsFor For example, example, the invention the invention provides provides for mixtures for mixtures of a of a TGFß TGF$ inhibitor, aa MEK/ERK receptor/ALK5inhibitor, receptor/ALK5 pathway MEK/ERK pathway inhibitor, RhoGTPase/ROCK inhibitor,a aRho GTPase/ROCK inhibitor, inhibitor, in in 25 particular 25 embodiments, particularembodiments, with with mammalian mammalian cells. For For cells. example, example, the the mixtures cancan mixtures be be included inin included cell culture cell culture media, withororwithout media, with cells.TheThe withoutcells. contents contents of cell of cell culture culture media media are generally are generally

art.Exemplary knownin inthetheart. known Exemplary cell cell culture culture mediamedia are described are described in detail in theinExamples. in detail the Examples. Generally, culturescomprising cell cultures Generally, cell comprising mammalian mammalian cells and and agents cellsagents of the of the invention invention (TGFß (TGF receptor/ALK5 inhibitor, aaMEK/ERK receptor/ALK5 inhibitor, pathway MEK/ERK pathway inhibitor, anda aRho inhibitor,and GTPase/ROCK RhoGTPase/ROCK 30 inhibitor) 30 willwill inhibitor) initially initially all all contain contain or or substantially allall substantially non-pluripotent non-pluripotent cells. cells. However, However, over over time, especially time, underthe especially under theconditions of of conditions thethe protocols protocols described described here,here, a portion a portion of the the cells ofcells will will become pluripotent become pluripotent (i.e.,iPSCs). (i.e., iPSCs).

44

Cells

[0168] Cells

[0168] be induced to induced to be to pluripotency can becan to pluripotency be cultured cultured according according to anyknown to any method method known in the in the art. art. General guidelinesforforculture General guidelines cultureconditions conditions to to generate generate iPSCs iPSCs canfound can be be found in, e.g., in, e.g.,

Maherali, etal., Maherali, et Cell Stem al., Cell Cell3:595-605 StemCell 3:595-605 (2008). (2008).

[0169] In some

[0169] In some embodiments, embodiments, theare the cells cells are cultured cultured in contact in contact withcells. with feeder cells. Exemplary feederExemplary 5 feeder 5 feeder cells cells include, but but include, are are not not limited limited to fibroblast to fibroblast cells, cells, e.g., e.g., mouse mouse embryonic embryonic fibroblast fibroblast

2022204080 cells.Methods (MEF)cells. (MEF) Methods of culturing cellscells of culturing on feeder cellscells on feeder are known in the in are known the art. art.

In some

[01701 In some

[0170] embodiments, embodiments, theare the cells cells are cultured cultured in the absence of feederofcells. in the absence cells. feederCells, Cells, for example, for canbebeattached example, can attached to to directly directly a solid surface culturesurface a solidculture (e.g.,a aculture (e.g., plate),e.g., cultureplate), via e.g., via a molecular a The tether.The moleculartether. inventors havehave inventors found that that found culturing cells cells culturing induced induced to pluripotency to pluripotency

10 havehave 10 a much a much greater greater efficiency efficiency of induction of induction to pluripotency (i.e., a (i.e., to pluripotency a greater greater of cellsof cells portion portion

achieve pluripotency)when achievepluripotency) the the when cells are are cells attached attached directly directly to the to the solid solid culturing culturing surface surface

compared compared thethe of of efficiency efficiency otherwise otherwise identically-treated thatthat cellscells identically-treated are are cultured cultured on feeder on feeder cells. cells.

Exemplary Exemplary molecular molecular tethers tethers include, include, but not but are not limited are limited to, Matrigel@, to, Matrigel®, an extracellular an extracellular

(ECM), matrix(ECM), matrix ECMECM analogs, analogs, laminin, laminin, fibronectin, fibronectin, or collagen. Those ofThose or collagen. of the skill in in skillart the art 15 however 15 however will recognize will recognize thatisthis that this is a non-limiting a non-limiting list and andother listthat that other molecules molecules can be can be used to used to attach to aa solid cells to attach cells solid surface. Methods surface. Methods forfor initial attachment initialattachment of of thethe tethers tethers to to thesolid the solid surface known are known surface are in in thethe art. art.

[0171] As described

[01711 As described herein, herein, in some in some embodiments, embodiments, the of the mixtures mixtures the invention invention of the can can include cellscells mammalian excludemammalian include oror exclude (including (including pluripotent pluripotent or non-pluripotent cells), cells), or non-pluripotent and oneand one 20 or or 20 more more a HDAC ofHDAC of a inhibitor, inhibitor, GSK3GSK3 inhibitor, or or inhibitor, an an L-type L-type CaCa channel channel agonist; activator agonist;ananactivator of of the the cAMP pathway;aaDNA cAMP pathway; DNAmethyltransferase (DNMT) methyltransferase(DNMT) inhibitor; inhibitor; a nuclear receptor a nuclearreceptor e.g., as ligand, e.g., ligand, as described described in PCTWO/2009/117439. in PCT WO/2009/117439.

VI. Kits VIII. Kits

[0172] The present invention

[0172] The present also provides also provides invention kits, e.g., kits, e.g., for use in generating forinusegenerating induced induced

25 stem stem pluripotent 25 pluripotent cells.cells. Such kits kitscomprise Suchcan can comprise anyoforthe any or all all reagents describeddescribed of the reagents herein, herein, including but not including but notlimited to:to: limited receptor/ALK5 inhibitor, a TGFßreceptor/ALK5 a TGFP inhibitor,a MEK/ERK pathway a MEK/ERK pathway

and/or aa Rho inhibitor, and/or inhibitor, GTPase/ROCK RhoGTPase/ROCK inhibitor, inhibitor, as described herein. herein. as described These These three threeoragents, agents, or thereof, can subsets thereof, subsets presentininthe canbebepresent kitininseparate thekit vials, orortogether separatevials, mixture.TheThe togetherasasa amixture. kitskits

of the of the invention invention can include,oneone alsoinclude, canalso or or more more of HDAC of an an HDAC inhibitor, inhibitor, GSK3 inhibitor, a GSK3 ainhibitor, or an or an Ca Ca L-type 30 L-type 30 channel channel agonist; an an agonist; activator the cAMP activatorofofthe pathway;a aDNA cAMP pathway; DNA methyltransferase methyltransferase

(DNMT) (DNMT) inhibitor; and and inhibitor; a nuclear a nuclear receptor receptor ligand. ligand.

45

In one

[0173] In one

[0173] embodiment, embodiment, theofkits the kits theof the invention invention will include will include one or one or more more types of types of mammalian mammalian (e.g., (e.g., human, human, mouse, mouse, rat, etc.) rat, etc.) cellscells and/or and/or cell cell culture culture media. media.

[0174] In aInparticular

[0174] a particular embodiment, embodiment, theof the kits kits the invention theofinvention will include one or one will include more or more polynucleotides comprising polynucleotides comprising expression expression cassettes cassettes of oneof for expression for expression orone moreorofmore Oct of Oct

5 polypeptide, 5 polypeptide, Klf polypeptide, a Klfa polypeptide, a Myc a Myc polypeptide, polypeptide, and a Soxand a Sox polypeptide. polypeptide. In addition,Inoraddition, or

2022204080 alternatively, the alternatively, the kits kits can can comprise oneorormore comprise one more isolated isolated transcription transcription factor factor proteins, proteins, e.g., e.g.,

one, two, one, three oror all two, three all four four of of an an Oct polypeptide,a Klf Octpolypeptide, a Klf polypeptide, polypeptide, a Myc a Myc polypeptide, polypeptide, and a and a polypeptide.In In Soxpolypeptide. Sox another another particular particular embodiment, embodiment, the transcription the transcription factor factor proteins proteins can be can be fused to fused to aa polypeptide sequence polypeptidesequence for for enhancing enhancing transport transport oftranscription of the factorfactor the transcription proteins proteins

10 across 10 across cellmembranes. cell membranes.

VI. VI. Usesforpluripotent Uses cells for pluripotent cells

[01751 TheThe

[0175] present present invention invention allowsallows for thefor study study the further further and development and development of stem of stem cell cell technologies, notnot includingbutbut technologies, including limited limited to,to, prophylactic prophylactic or therapeutic or therapeutic uses. uses. For example, For example, in in embodiments, someembodiments, some cellscells of the of the invention invention (either (either pluripotent pluripotent cells cells or cells or cells induced induced to to 15 15 differentiate differentiate along along a desired a desired cell cell fate)fate) are are introduced introduced into individuals into individuals in thereof, in need need thereof, including butnot includingbut notlimited to,individuals limitedto, needof of individualsininneed regeneration regeneration of organ, of an an organ, tissue, tissue, or cell or cell

type. Insome type. In embodiments, someembodiments, the cells the cells are originally are originally obtained obtained in a biopsy in a biopsy from anfrom an individual; individual;

inducedinto induced intopluripotency pluripotencyas as described described herein, herein, optionally optionally induced induced to differentiate to differentiate (for (for intoa aparticular examplesinto examples particulardesired desiredprogenitor progenitor cell) cell) andand thenthen transplanted transplanted back back into into the the 20 individual. 20 individual. In some In some embodiments, the cellsthe embodiments, are cells are genetically genetically modified modified prior prior to their to their the individual. into the introduction into introduction individual.

In some

[01761 In some

[0176] embodiments, embodiments, the pluripotent the pluripotent cells generated cells generated accordingaccording to theofmethods to the methods of the invention the inventionare aresubsequently subsequently induced induced to form, to form, for example, for example, hematopoietic hematopoietic (stem/progenitor) (stem/progenitor)

neural (stem/progenitor) cells, neural cells, cells(and (stem/progenitor) cells (andoptionally, more optionally,more differentiated differentiated cells, cells, such such as subtype as subtype

specific 25 specific 25 neurons, neurons, oligodendrocytes, oligodendrocytes, etc), pancreatic etc), pancreatic cells (e.g., cells (e.g., endocrine endocrine progenitor progenitor cell or cell or pancreatic hormone-expressing pancreatic hormone-expressing cells), cells), hepatocytes, hepatocytes, cardiovascular cardiovascular (stem/progenitor) (stem/progenitor) cells cells (e.g., cardiomyocytes, (e.g., endothelialcells, cardiomyocytes, endothelial cells,smooth smooth muscle muscle cells), cells), retinal retinal cells, cells, etc. etc.

A variety

[0177] A variety

[0177] of methods of methods are known are known for inducing for inducing differentiation differentiation of pluripotent of pluripotent stem stem cells into cells into desired cell types. desired cell types. AAnon-limiting non-limiting listofofrecent list recentpatent patentpublications publications describing describing

30 methods 30 methods for inducing for inducing of stemof differentiation differentiation steminto cells cells into various various cell fates fates follows: cellfollows: U.S. U.S. Patent Patent Publication Nos.: Publication Nos.: 2007/0281355; 2007/0264709; 2007/0269412;2007/0264709; 2007/0281355; 2007/0269412; 2007/0259423; 2007/0259423;

2007/0254359;2007/0196919; 2007/0254359; 2007/0172946; 2007/0196919;2007/0172946; 2007/0141703; 2007/0141703; 2007/0134215. 2007/0134215.

46

[0178] A variety of diseases may be ameliorated by introduction, and optionally by introduction, targeting, and optionally targeting,

[01781 A variety of diseases may be ameliorated of pluripotent of pluripotent cells of the cells of inventiontoto aa particular the invention injuredtissue. particular injured Examples tissue. Examples of disease of disease

resulting fromtissue resulting from tissueinjury injuryinclude, include,but butare arenot notlimited limitedto,to,neurodegeneration neurodegeneration disease, disease,

cerebral obstructivevascular infarction, obstructive cerebral infarction, myocardial disease,myocardial vasculardisease, infarction, infarction, cardiac cardiac failure, failure,

5 chronic 5 chronic obstructive lung lung obstructive disease, disease, pulmonary pulmonary emphysema, interstitialinterstitial bronchitis, bronchitis, emphysema, pulmonary pulmonary disease, asthma,hepatitis disease, asthma, (liverdamage), hepatitisBB(liver damage), hepatitis hepatitis C (liver C (liver damage), damage), alcoholic alcoholic hepatitis hepatitis 2022204080

(liver damage), (liver hepaticcirrhosis damage), hepatic (liverdamage), cirrhosis(liver damage), hepatic hepatic insufficiency insufficiency (liver (liver damage), damage),

pancreatitis, diabetes pancreatitis, mellitus, Crohn diabetes mellitus, disease,inflammatory Crohndisease, inflammatory colitis, IgA IgA colitis, glomerulonephritis, glomerulonephritis,

glomerulonephritis, renalinsufficiency, glomerulonephritis,renal decubitus, insufficiency,decubitus, burn, burn, sutural sutural wound, wound, laceration, laceration, incised incised

wound, 10 wound, 10 bite wound, bite wound, dermatitis, dermatitis, cicatricial keloid,keloid, keloid,keloid, cicatricial diabetic ulcer, ulcer, diabetic arterial ulcer,ulcer, arterial and and ulcer. venousulcer. venous

[0179] In one embodiment, iPSCs can

[0179] In one embodiment, be used iPSCs can in be various used in assays andassays various screenand screen to to identify identify thatmodulate moleculesthat molecules modulate their their function, function, including including but not not limited but limited to promoting to promoting iPSC survival iPSC survival

and/or differentiation. and/or differentiation.

15 15 EXAMPLES EXAMPLES

[0180] The The

[0180] following following examples examples are offered are offered to illustrate, to not but notbut to illustrate, to the limit the claimed limitclaimed invention. invention.

Example1:1:A Achemical Example chemicalplatform forfor platform improved improved induction induction of human iPSCsiPSCs of human

[0181] Mesenchymal

[0181] Mesenchymal typetype fibroblasts fibroblasts reprogrammed reprogrammed withwith the the "four-factors"(OCT4, "four-factors" (OCT4, SOX2, SOX2,

20 KLF4KLF4 20 & c-MYC; & c-MYC; 4TFs hereafter) 4TFs hereafter) underwent underwent dramatic dramatic morphological changeschanges morphological that resulted that resulted in in iPSCswith iPSCs withdistinct cell polarity, distinctcell boundaries polarity, boundaries andand cell-cell interactions.TheThe cell-cellinteractions. reprogrammed reprogrammed

cells expressed cells E-cadherin,a marker expressed E-cadherin, a marker for for epithelial epithelial cells cells (Hay, (Hay, E.D., E.D., ActaActa Anat.Anat. (Basel) (Basel) 154, 154, 8-20 (1995)), 8-20 which (1995)), which is is alsohighly also highly expressed expressed in human in human embryonic embryonic stem stem cells cells (hESCs). (hESCs). We We reasoned thatpromote factorsthat thatfactors reasonedthat promotethethe mesenchymal mesenchymal to epithelial to epithelial transition such as such (MET),(MET), transition as 25 TGFßTGFp 25 pathway pathway antagonists, antagonists, would have have would a direct a direct impact impact on the on the reprogramming reprogramming process. process. In In addition, MEK-ERK addition, pathway MEK-ERK pathway inhibitionwaswas inhibition previouslyshown previously to to shown playananimportant play importantrole in role in ofreprogramming steps of various steps various reprogramming(Chen, S. et S. (Chen, et al., al., Proc.Proc. Natl. Natl. Acad. Acad. Sci.104, Sci. USA USA 104, 10482-87 10482-87 (2007); Y.etet al., Shi, Y. (2007); Shi, al., Cell Cell Stem Stem Cell 525-8(2008)). Cell2,2, 525-8 Furthermore, (2008)).Furthermore, factors factors promoting promoting cell cell couldalso survival could survival beneficialininimproving alsobebebeneficial improving reprogramming reprogramming efficiency. efficiency. Consequently, Consequently, we we 30 focused 30 focused on small on small molecules that canthat molecules can regulate regulate these these three three processes processes and as and pathways, pathways, small as small have moleculeshave molecules many many (Feng,(Feng, advantages advantages B. etCell B. et al., al., Stem Stem4,Cell CellCell 301-12 (2009); (2009); 4, 301-12 Shi, Shi, Y. et Y. et al., Cell al., Cell Stem Cell 2, Stem Cell 2, 525-8 (2008);Xu,Xu, 525-8 (2008); et et Y. Y. al.,Nature al., Nature 453, 453, 338-44 338-44 (2008)) (2008)) in studying in studying

biological processesand biological processes choice a saferchoice andarearea safer than than genetic genetic manipulation. manipulation. Here Here we we describe describe a a

47

simplechemical simple platform chemicalplatform that that substantially substantially enhances enhances generation generation of fully of fully reprogrammed reprogrammed

fromfrom iPSCs humaniPSCs human fibroblasts fibroblasts through much and a mucha faster through faster and more efficient process. process. more efficient

[0182]

[0182] WeWe tested testedknown inhibitors known inhibitorsofofthe the TGFß TGFpreceptor receptorand andMEK MEK on Ix10 on 1x10 4 (Feng, (Feng, B. B. et et Cell Stem al., Cell al., 4, 301-12 Cell 4, Stem Cell 301-12(2009)) human (2009))human primary primary fibroblasts fibroblasts (CRL2097 (CRL2097 or BJ) that werethat or BJ) were 5 retrovirally 5 retrovirally transduced with with transduced the 4TFs, the 4TFs, for their effecteffect for their on reprogramming kinetics kinetics on reprogramming and and 2022204080 efficiency (see efficiency (see Fig. Fig. la 1afor for details). details). On Onday day7 (D7) 7 (D7) post-infection, post-infection, the the compounds compounds were were added, added, individually thethe combinations,andand individually ororinin combinations, cultures cultures were were examined examined for iPSCs iPSCs for over nextthe theover 1-3next 1-3 weeks. weeks.

On 7day

[01831 On day

[0183] post-treatment (D14),(D14), 7 post-treatment we observed we observed the strongest effect in effect the strongest in the the cultures cultures 10 treatedwith 10 treated ALK5 combinationofofALK5 witha acombination inhibitor SB431542 inhibitorSB431542 (2 pM) (2 µM) MEK MEK and and inhibitor inhibitor

PD0325901 PD0325901 (0.5(0.5 whichwhich µM),pM), resulted in ~45 in resulted large large -45 ALP colonies colonies ALP+ (Fig. 1b) with I b) with characteristic (Fig.characteristic morphology,ofofwhich hESC-like morphology, hESC-like whichover were TRA-1-81 colonies were over2424colonies andabout I d),and +(Fig.1d), TRA-1-81(Fig. 6-10 about6-10 colonies stainedpositive colonies stained forSSEA4 positivefor SSEA4and and NANOG, NANOG, mature pluripotency a mature apluripotency is notthat factor thatfactor is not (Fig.le Iand introduced(Fig. ectopically introduced ectopically e and 1f). Moreover, I f).Moreover, the treated the treated cultures cultures showed showed high high level level 15 of of expression 15 expression endogenous endogenous mRNA mRNA for the the pluripotency for pluripotency genes genes (Fig. (Fig. Ic).In In 1c). contrast, no contrast, no NANOG* colonies NANOG+ colonies were observed were observed in the untreated in the untreated control cultures & Fig. le (Fig. le (Fig. control cultures &or 4a) in 4a) or in Fig. that were cultures that cultures treated with were treated PD0325901 withPD0325901 alone alone (Fig. (Fig. 4a). However, 4a). However, in the cultures in the cultures treated treated with only with onlySB431542, SB431542, we still we still observed observed 1-2hESC-like 1-2 ALP ALP+ hESC-like colonies (Fig. 4a). (Fig. colonies Importantly, 4a). Importantly, combined the combined the effect of of effect both thethe both inhibitors inhibitors (Fig. (Fig. 4b 4b & 4c), & 4c), as well as well as the as the individual individual effect effect of of 20 SB431542 20 waswas SB431542 dose dose dependent. dependent.

[0184]

[01841 When we maintained When the the we maintained SB431542 plus plus SB431542 PD0325901 treated PD0325901 cultures cultures treated for days for 30 30 days without weobtained splitting, we withoutsplitting, obtainedabout iPSCiPSC 135135 about colonies colonies per well well 2d), per (Fig. >100a fold (Fig.a2d), >100 fold improvement improvement overover in efficiency in efficiency the the conventional conventional method. method. Consistent Consistent with previous previous with reports reports (Takahashi,K.K.etetal., (Takahashi, Cell131, al., Cell 131, 861-72 in in (2007)), 861-72(2007)), untreated untreated controls controls carrying 4TFs,4TFs, carrying we we observed 25 observed 25 iPSC colonies 1-2 colonies 1-2 iPSC in addition in addition to several to several granulate granulate These2c). (Fig. 2c).(Fig. colonies colonies These granulate structures granulate structureshave havebeen been suggested suggested to partially to be be partially reprogrammed reprogrammed coloniescolonies (Takahashi, (Takahashi,

al., Cell et al., K. et K. 131, 861-72 Cell 131, 861-72 (2007)). alsoalso (2007)).We We observed observed granulate granulate colonies in the in colonies the SB431542 SB431542

treated treated cultures, outnumbered whichoutnumbered cultures, which by several by several foldfew fold the few hESC-like thehESC-like colonies. colonies.

Interestingly, numberofof the number Interestingly, the granulate granulate colonies was was colonies dramatically dramatically reduced in the in reduced the combined combined

SB431542 30 SB431542 30 and PD0325901 and PD0325901 treatment, which which treatment, resulted resulted in a concomitant in a concomitant increase increase in the in the number number

of hESC-like of hESC-like colonies. thata acombined suggested that colonies. This suggested inhibitionofof combined inhibition ALK5 and ALK5 and MEK may MEK may

guide partially guide reprogrammed partially reprogrammed colonies to a to colonies a fully fully reprogrammed reprogrammed state state and and improve thereby improve thereby the the overall reprogramming overall reprogramming process. process. Moreover, Moreover, the fact factwethat thethat we observed observed improved ofinduction improved induction of iPSCs earlyasas7 7days iPSCsasasearly post-treatment dayspost-treatment suggests that that suggests treatment these these with with treatment small small molecules molecules

48 not not only improved only improved thethe efficiency efficiency of the of the reprogramming but may but processprocess reprogramming havealso have alsomay accelerated (Fig. 1a). kinetics (Fig. its kinetics accelerated its Additionalexperiments la). Additional are are experiments required required to determine to determine whether whether 2022204080 10 Jun the reprogrammed the reprogrammed cells cells at this'stage at this indeed stage indeed become fully fully become independent independent of exogenous of exogenous reprogramming reprogramming factors factors earlier earlier than than in untreated in untreated cultures. cultures.

[01851 55 [0185] Although Although iPSC colonies were were iPSC colonies picked picked and expanded, as in as and expanded, in hESC hESC cultures, cultures, the the cultures split cultures by trypsinization split by resulted inin poor trypsinization resulted poorsurvival. From survival.From a recent a recent screen screen performed performed in in laboratoryweweidentified our laboratory our a novel identifieda novel small small molecule, molecule, Thiazovivin Thiazovivin (Fig. 5), 5), which (Fig.which dramatically dramatically

survivalof of improvedthethesurvival improved upon upon hESCs hESCs trypsinization. trypsinization. Addition Addition of Thiazovivin of Thiazovivin to our to our cocktail cocktail of SB431542 of SB431542 and and PD0325901 PD0325901 also vastly vastly improved also improved the of the survival survival of iPSCs iPSCs after splitting splitting after by by 10 trypsinization 10 2a), and (Fig. 2a), trypsinization(Fig. largenumber and aa large ofreprogrammed number of colonies were reprogrammed colonies were obtained. From obtained. From 10,000 cells that 10,000 cells wereoriginally that were originallyseeded, a single1:41:4 seeded,a single splittingonondayday splitting 14 resulted 14 resulted in -1,000 in ~1,000

on on colonies hESC-likecolonies hESC-like dayday 30 (Fig. 2e),2e), 30 (Fig. while while two rounds two rounds of splitting of splitting (on14day (on day and 14 on and day on day 21 (1:10)) 21 resultedinin ~11,000 (1:10)) resulted hESC-like -11,000hESC-like (Fig.(Fig. colonies colonies 2e)& 2c & 2c dayon on2e) 30.day 30. colonies These These colonies showed high showed high levels levels of of mRNA mRNA endogenous endogenous (Fig. 2f)(Fig. and protein (Fig. 2b & (Fig. protein expression 2f) and expression 2b & 2c) of 2c) of

pluripotency 15 pluripotency 15 while while markers, markers, the expression the expression of the of the four four transgenes transgenes could hardly detectedbe be hardly could detected (Fig. 2f). (Fig. contrast, no In contrast, 2f). In iPSCcolonies no iPSC were colonieswere obtained obtained from from untreated untreated or 2 compound-treated or 2 compound-treated

samples thatwere samplesthat trypsinized weretrypsinized (Table (Table 1). 1).

Table 1: Table the number comparisonofofthe 1: AA comparison iPSCcolonies numberofofiPSC colonies observed untreated, 22compound in untreated, observed in compound

treated and 33 compound treated and compound treated treated cultures cultures on 30. on day day 30.

Nocompound No compound 2compounds 2 compounds 3compounds 3 compounds

No splitting No splitting 11 135 135 205 205

splitting (on 1 splitting 1 (on day 14) day 14) 00 00 900 900

2 (on day splitting (on 2 splitting day 14 day21)21) 14&&day N/A N/A N/A N/A 11,000 11,000

20 20

[0186] To examine

[01861 To examine whether whether the positive the positive effect effect of Thiazovivin is solelyisdue of Thiazovivin solely due to survival to survival of of after splitting colonies after colonies whether itit also or whether splitting or augments also augments thethe reprogramming effect effect reprogramming of combined of combined

andPD0325901 SB431542and SB431542 PD0325901 treatment, we we treatment, tested tested the3 3compound the compoundcocktail on on cocktail 4TF-transduced 4TF-transduced

cells that were cells that were not splitting. InInthese subjectedtotosplitting. not subjected day14 14 cultures,bybyday thesecultures, we we observed observed -25 ~25 large large colonies 25 colonies 25 wereallallexpressing thatwere that expressing Nanog le). By (Fig. 1e). Nanog(Fig. day30 By day 30we weobserved verylarge -205 very observed~205 large NANOG+ colonies NANOG* (Fig. colonies (Fig.2d), 2d),that also TRA-1-81 were also that were +andSSEA4 TRA-1-81 and (data(data SSEA4+ not shown), not shown), which which

translated to translated to aa more than200 more than 200 fold fold improvement improvement in efficiency in efficiency over over no no compound compound treatment,treatment,

and aa two-fold and two-foldincrease over increaseover 2 compound 2 compound treatment. treatment.

49

[0187] TwoTwo

[0187] compound compound treatment treatment also resulted also resulted in anumber in a larger alkaline phosphatase alkalineofphosphatase- largerofnumber compared coloniescompared positive colonies positive to untreated to untreated controls when when controls the reprogramming the reprogramming factors factors were were usinga alentiviral, introducedusing introduced thanaaretroviral rather than lentiviral, rather retroviral system (Fig.6a). system(Fig. 6a).Furthermore, the the Furthermore, 3 3 compound compound cocktail did did cocktail not not appear appear to influence to influence reprogramming reprogramming factor expression factor expression from from 55 retroviral retroviral vectors vectors (Fig. (Fig. 6b-f). 6b-f).

2022204080 [01881 TheThe

[0188] iPSC iPSC colonies generatedusing coloniesgenerated the33 compound usingthe compoundcocktail werereadily cocktailwere readily and stably and stably long expandedforforlong expanded term term under under conventional conventional hESC culture hESC culture conditions (over 20 (over conditions 20 passages) passages) and and they closely they resembled closely resembled hESCs hESCs in terms in terms of morphology, typical typical of morphology, pluripotency pluripotency marker marker expression anddifferentiation expression and differentiationpotentials. They potentials.They exhibited exhibited a normal a normal karyotype karyotype (Fig. 7) and 7) and (Fig. could 10 could 10 into into be differentiated be differentiated derivatives derivatives ofthree of all germ germ all three layers, layers, both both in vitro (Fig. (Fig. in vitro 3a 3a & 3b) & 3b) and inin vivo and (Fig. 3c). vivo (Fig. Theseresults 3c). These thatthat suggested alsosuggested resultsalso there no no is is there short short term term adverse adverse effect effect

themuch withthe associatedwith associated much more more convenient convenient trypsinization trypsinization procedure. procedure.

The

[01891 The

[0189] demonstration demonstration that TGFandand thatTGFß MEK-ERK MEK-ERK pathway pathway inhibition inhibition improved improved

reprogramming fibroblast reprogramming fibroblast suggested suggested critical rolesroles critical for these two two for these signaling signaling pathways and MET and pathways MET 15 in the in mechanisms 15 mechanisms the process. process. Consistently, addition addition Consistently, of TGFp of TGFß had an inhibitory inhibitory had aneffect on 4 effect on 4 reprogramming factor-mediatedreprogramming factor-mediated of fibroblasts (data (data of fibroblasts not shown). TGFß andTGFp not shown). and its its family family members members play play important important contextual roles roles contextual in self-renewal in self-renewal and differentiation of ESCsof ESCs and differentiation andMiyazono, (Watabe,T.T.and (Watabe, Miyazono, K., Cell K., Cell Res. Res. 19, 103-15 19, 103-15 (2009)). (2009)). Moreover, TGFß is a TGFp Moreover, is a prototypical of of induction cytokineforforinduction prototypical cytokine epithelial mesenchymal epithelialmesenchymal transition (EMT) (EMT) transition and and 20 maintenance 20 maintenance of the of the statestate mesenchymal mesenchymal (Willis, B.C.and (Willis,B.C. Borok,Z., andBorok, Physiol. Lung Am.J.J. Physiol. Z.,Am. Cell Lung Cell

Mol. Physiol.293, Mol.Physiol.293, L525-34 L525-34 (2007)). (2007)). A major A major endofpoint end point this of signaling, in this in this signaling, this context, context, is is downregulation down of of regulation E-cadherin E-cadherin (Thiery, J.P. J.P. (Thiery, and Sleeman, and Sleeman, J.P., Nat. Rev. Cell Nat. Mol. J.P., Rev. Cell Biol., Mol.Biol., 7, 7, 131-42 has has E-cadherin (2006)).E-cadherin 131-42(2006)). shownshown been been to be important for the for to be important the maintenance maintenance of of pluripotency ofof pluripotency ESCs has has andand ESCs beenbeen recently recently suggested suggested be a regulator to be atoregulator ofNANOG of NANOG

expression 25 expression 25 (Chou, Y.F. etY.F. (Chou, al., 135, al.,etCell Cell449-61 (2008)).(2008)). 135, 449-61 inhibitioninhibition ThereforeTherefore of TGFß of TGFP signaling, which signaling, resultsininde-repression whichresults de-repressionof of epithelial fate,could epithelialfate, couldbenefit thethe benefit reprogramming reprogramming

processinin multiple process multipleways. ways.ERKERK signaling signaling also promotes also promotes EMT J.P. EMT (Thiery, (Thiery, J.P. andJ.P., and Sleeman, Sleeman, J.P. Nat. Nat. Rev. Mol.Cell Rev. Mol. Biol.7,7,131-42 CellBiol. 131-42 (2006)), and and (2006)), is downstream is downstream ofinTGFO of TGFß in the process the process

Y.F.etetal., (Chou,Y.F. (Chou, al., Cell 135, 449-61 Cell135, (2008)).We had 449-61(2008)). had previously Wepreviously shown that the that shown theofeffect effect of reversine, 30 reversine, 30 a small a small molecule molecule which which can reprogram myoblasts myoblasts can reprogram to a multipotent to a multipotent state, is state, is mediatedininpart mediated through partthrough of of inhibition inhibition MEK-ERK MEK-ERK (Chen, (Chen, S. et al., et al.,Natl. S. Proc. Proc.Acad. Acad Natl.Sci. US Sci. US A 104, A 104, 10482-87 mayexplain This may (2007)). This 10482-87 (2007)). explain the observed in effect observed the effect inreprogramming reprogramming when it when it combinedwith was combined was TGFp withTGF inhibition. inhibition.

50

[0190] The The

[0190] chemical chemical platform platform described here is here described unique, in that in is unique, it that modulates upstream upstream it modulates pathways signaling pathways signaling andand could could radically radically improve improve reprogramming reprogramming on acell on a general type,cell general liketype, like fibroblasts. The fibroblasts. conditions chemicalconditions The chemical described herehere described provide provide a basic a basic platform platform for non-viral for non-viral

and non-DNA and non-DNA based based (Zhou, (Zhou, al.,etCell H. et H. Stem Stem al., Cell Cell Cell 4, 4, 381-84, 381-84, (2009)), (2009)), more efficient more efficient and and 5 safer 5 safer reprogramming reprogramming methods, methods, which which could could yield yield an an unlimited unlimited supply supply of of safehuman safe human iPSCs iPSCs

for various for applications. various applications. 2022204080

METHODS METHODS Cell culture Cell culture

[0191]

[01911 Primary skin Primary CRL2097 fibroblasts CRL2097 skinfibroblasts and BJ BJ and (neonatalforeskin) (neonatal were purchased foreskin) were from purchasedfrom 10 ATCC. ATCC. All cell All cell culture culture media media reagents reagents were were purchased purchased from from Invitrogen Invitrogen Corporation,CA.CA. Corporation,

The cells The maintained in were maintained cells were in DMEM DMEM (10313-021) containing10%10% (10313-021)containing FBSFBS (10439-024), (10439-024), 1X IX MEM MEM Non-Essential Non-Essential Amino Amino acidacid (11140-050), (11140-050), IX Glutamax 1X Glutamax (35050-061), mM 10 mM10Hepes (35050-061), Hepes and 0.1 (15630-080) and (15630-080) 0.11mM 2-Mercaptoethanol(21985-023). 1mM 2-Mercaptoethanol Cellswere (21985-023).Cells passaged werepassaged using 1:51:5using

(iX)trypsin-EDTA 0.05%(1X) 0.05% (25300-054). trypsin-EDTA(25300-054).

15 Plasmids 15 Plasmids

ThepMXs

[01921 The

[0192] encoding the vector encoding pMXsvector thehuman cDNAs for humancDNAs forOCT4, SOX2, c-MYC OCT4,SOX2, and c-MYC and KLF4, described KLF4,described before before (Takahashi, K. etK. (Takahashi, al., CellCell et al., 131, 131, 861-72 861-72 (2007)), (2007)), were obtained were obtained from from ADDGENE. ADDGENE. Mouse Mouse Slc7al Slc7al ORF was was cloned ORFcloned into pWPXLD into pWPXLD (Addgene), (Addgene), as described as described

previously (Takahashi, previously(Takahashi, K. K. et et al.,Cell al., Cell131, 861-72 131,861-72 (2007)). (2007)).

20 Retroviral 20 Retroviral Infection iPS iPS andand Infection Cell Cell Generation Generation

[0193] OCT4,NANOG, carrying OCT4, Lentivirusescarrying

[01931 Lentiviruses SOX2SOX2 NANOG, & LIN28 & LIN28 were produced were produced as described as described

before before (Yu, al., Science (Yu,J.J.etet al., 318, 1917-20 Science 318, 1917-20(2007)). For For (2007)). retrovirus retrovirus production, PLAT-EPLAT-E production, packaging cells were plated at at 6 cells/well After After 24 hours, the cells packaging cells were plated 1x10 cells/well 1x10 of a of a 6-well 6-well plate. plate. 24 hours, the cells

were were transfected withpMXs transfected with pMXs vectors carrying OCT4, vectors carrying OCT4, SOX2, c-MYC SOX2,c-MYC andand KLF4 KLF4 cDNAs using using cDNAs 25 Fugene 25 Fugene 6 transfectionreagent 6 transfection accordingtotomanufacturer's (Roche)according reagent(Roche) instructions. Twenty-four manufacturer'sinstructions. Twenty-four

hours after transfection, hoursafter medium the medium transfection, the waswas replaced fresh fresh with with replaced and the and mediummedium wasplate platethe was transferred to 32 transferred to forretrovirus 32 °C°Cfor production. retrovirusproduction. The The viruses were were viruses collected at 48 at collected hours and 72 and 72 48 hours hours, and hours, filtered with andfiltered 0.45µmpm with0.45 filterbefore filter before transduction. transduction.

[0194] The Slc7al-expressing human fibroblast cells were

[01941 The Slc7al-expressing human fibroblast seeded cells at seeded were 1x10 cells/well at xI105 ofcells/well a 6 of a 6 30 wellwell 30 plate plate on day on the the 1. 0.252,ml0.25 2, day dayOn 1.dayOn ml of of each each retroviral retroviral supernatant was added was supernatant added to the to the

cells in cells in the the presence of66 µg/ml presence of polybrene. pg/mlpolybrene. A second round round A second of transduction was donewas of transduction done on day on day 3. Infection 3. Infection efficiency was efficiencywas estimated estimated by fluorescence by fluorescence microscopy on cellson microscopy cells transduced transduced in in

51

parallel with parallel GFPororRFPRFP with GFP gene-carrying gene-carrying retroviruses. Seven Seven retroviruses. days after after initial daysinitial transduction, transduction,

4 of a 6ofwell plate plate fibroblasts were harvested by by fibroblasts were harvested trypsinization andand trypsinization re-plated at 1x10 re-plated at 1x10 cells/well cells/well a 6 well coated with coated matrigel(1:50 withmatrigel (1:50dilution, 354234, dilution,catcat354234, BD Biosciences). BD Biosciences). For compound For compound treatment,treatment,

the cells the cellswere werecultured culturedin in human reprogramming medium humanreprogramming (DMEM/F12, medium (DMEM/F12, 20% 20% Knockout Knockout

5 serum 5 serum replacer,1x lxMEMMEM replacer, Non-Essential Non-Essential amino acid,acid, amino 1x glutamax, 0.110.11 1x glutamax, mM mM 2- 2 20 ng/ml Mercaptoethanol, 20 Mercaptoethanol, ng/ml bFGF and1,000 bFGFand U/mlLIF) 1,000U/ml andwere LIF)and weretreated with 22 µMM treated with 2022204080

0.5µMpMPD0325901 (Stemgent),0.5 SB431542(Stemgent), SB431542 PD0325901 (Stemgent), 0.5 0.5 (Stemgent), M Thiazovivin, µM Thiazovivin, or or combinations of the combinations of the compounds. Themedia compounds. The werechanged mediawere changed every 2-32-3 every daysdepending days on on depending the the

cell density. cell days Sevendays density. Seven after after compound compound treatment, eithereither treatment, the plates the plates were and were fixed fixed and stained stained

10 for for 10 Alkaline Alkaline phosphatase phosphatase (ALP) activity, (ALP) activity, or stained or stained for protein for protein markers,markers, or the cultures or the cultures were were continuedwith continued without withororwithout indicated indicated splitting splitting by by trypsinization tilltill trypsinization dayday 30. 30. For For split split cultures, cultures,

cells were the cells the split (1:4) were split and re-plated (1:4) and ontoirradiated re-plated onto CF-I irradiatedCF-1 MEFMEF feeder layerlayer feeder 105x (2.5 X(2.5 105 cells/well) in each cells/well) in well ofof66 well each well plateand wellplate were andwere split again (1:10)again split(1:10) dayday on on 21. 21. The cells The cells were were maintainedininthe maintained same thesame and and media media compound cocktailcocktail compound described described above except forexcept above the for the 15 concentrationsof of 15 concentrations (0.5(0.5 PD0325901 PD0325901 for D14 pM D14 µM for 1 µM1 for and and for D21) M D21) and SB431542 (0.5-1 (0.5-1 and SB431542 µM pM after D14). The after D14). iPSC colonies The iPSC subsequently maintained colonies were subsequently maintained in conventional hESC in conventional hESC media in media in absence of the absence the of the theabove abovecompounds. compounds.

Alkaline Phosphatase Alkaline Stainingand PhosphataseStaining andImmunocytochemistry Immunocytochemistry

Alkaline

[0195] Alkaline

[0195] phosphatase phosphatase staining staining was performed using ALPusing was performed ALPkit detection (cat no:kit detection (cat no: 20 SCR004, 20 SCR004, Chemicon) Chemicon) according the product to theto product according instructions. ForFor instructions. immunocytochemistry, immunocytochemistry, cells cells

fixed in were fixed were in 4% 4% paraformaldehyde min, RT), (10 min, paraformaldehyde (10 RT), washed twice with washedtwice PBS,blocked with PBS, blockedusing 5% using 5% donkeyserum normal donkey normal (Chemicon) serum(Chemicon) and and 0.1% 0.1% TritonX-100 (15 (15 TritonX-100 min, min, and and RT)RT) thenthen treated with treatedwith primary antibodies primary °C.The overnight atat4 4°C. antibodies overnight Theprimary antibodiesused primaryantibodies wereanti-NANOG usedwere (cat anti-NANOG (cat

no: AB9220, no: AB9220, Chemicon, Chemicon, 1:1,000); 1:1,000); anti-OCT4 anti-OCT4 (cat no: (cat no: sc-5279, sc-5279, Santa Santa Cruz Cruz1:200), biotech, biotech, 1:200), 25 anti-SSEA 25 anti-SSEA 4 (cat no:no: 4 (cat mab4304, mab4304, Chemicon, Chemicon, 1:500), 1:500), anti-Tra-1-81 anti-Tra-1-81 (cat (cat 560123,BD BD 560123,

Biosciences, anti-Tra-1-81(mAb 1:100),anti-Tra-1-81 Biosciences, 1:100), (mAb 4381, 4381, Chemicon, anti-pIl TUBULIN 1:500), anti-ßIII Chemicon,1:500), (cat TUBULIN (cat

no: MMS-435P, no: Covance MMS-435P, Covance Research Research Products Products Inc, Inc, 1:1000), anti-PDX 1:1000),anti-PDX 1 (1:500) kindgift 1 (1:500)(a(akind gift from Wright), anti-BRACHYURY Dr. C. Wright), from Dr. No: No: anti-BRACHYURY (cat (cat AF2085, AF2085, R&D, final final concentration R&D, concentration 0.2 0.2 The pg/ml). The µg/ml). cellswere cells were washed twicetwice washed withandPBS with PBS thenand then with treated treated secondary antibodies antibodies with secondary 30 forfor 30 1 hour 1 hour atatroom temperature.TheThe roomtemperature. secondary secondary antibodies usedwere antibodiesused wereAlexa Alexa fluor488 fluor 488donkey donkey anti-rabbit or anti-mouse anti-rabbit or IgG anti-mouse IgG (Invitrogen, (Invitrogen, 1:1,000) AlexaAlexa and and 1:1,000) fluor fluor 555 donkey 555 donkey anti-rabbit anti-rabbit or or anti-mouseIgG1gG anti-mouse (Invitrogen, (Invitrogen, 1:1,000). 1:1,000). Nuclei Nuclei were stained were stained with with 0.5 µg/ml g/ml 0.5DAPI DAPI (Sigma). (Sigma). Images were Images using aa Nikon captured using werecaptured Eclipse TE2000-U/X-cite Nikon Eclipse 120EXFO TE2000-U/X-cite 120 EXFOmicroscope withwith microscope a a photometric CoolSnap photometric CoolSnapHQ2 HQ2 camera. camera.

52

In vitro In Differentiationand vitroDifferentiation andTeratoma Assay Teratoma Assay

Generation

[01961 Generation

[0196] of embryoid bodies bodies of embryoid and in vitro vitro differentiation and indifferentiation were performed were performed as as describedelsewhere described elsewhere (Takahashi, (Takahashi, K.al., K. et et al., CellCell 131,131, 861-72 861-72 (2007)). (2007)). Forteratoma For the the teratoma assay, assay, 3-5 cells were millioncells 3-5 million under injectedunder wereinjected thethe kidney kidney capsule capsule of SCID of SCID mice. mice. Thirty one daysone Thirty days later later 55 the the were were tumors tumors excised excised and infixed and fixed in 4% paraformaldehyde 4% paraformaldehyde and histologically and histologically analyzed analyzed at the at the

2022204080 TSRI histology core TSRI histology facility.The core facility. useofof Theuse SCIDmice SCID approved by was approved micewas the UCSD by the animal UCSD animal

committee. research committee. research

RT-PCR RT-PCR

[0197] RNA TotalRNA

[0197] Total waswas extracted extracted from from cells usingRNeasy cellsusing RNeasyminikit (Qiagen).cDNAs minikit(Qiagen). werewere cDNAs synthesized 10 synthesized 10 according according to product to product instructions instructions using superscript using superscript III first first strand IIIstrand synthesis synthesis kit kit Two (Invitrogen). Two (Invitrogen). microliters microliters of the of the reaction reaction product product was for was used for 24-28 used24-28 PCR cycles cycles PCRusing using primers.TheThe respective primers. respective sequences sequences of the the primers of primers are described are described elsewhere elsewhere (Takahashi, (Takahashi, K. et K. et Cell 131, al., Cell al., 131, 861-72 (2007)). 861-72 (2007)).

Flow cytometry Flow cytometry

15 [01981 15 [0198] For flow For flow cytometry cytometry analysis, the the analysis, cultures weremildly cultureswere trypsinizedand mildlytrypsinized harvested andharvested from 66 well from plates.The well plates. The cells cellswere washed and werewashed resuspended in andresuspended inFACS FACS buffer (PBS, 22 mM buffer (PBS, mM

EDTA, 2 2mMmM EDTA, HEPES, HEPES, 1% FBS), 1% FBS), and were were analyzed and analyzed on a FACS CaliburCalibur on a FACS cytometer cytometer (Becton (Becton

San Dickinson,San Dickinson, CA)CA) Jose, Jose, withwith the CellQuest the CellQuest program. program.

SynthesisofofN-(cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinolin- Example2:2:Synthesis Example N-(cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinolin 1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide 20 1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide 20 (Thiazovivin) (Thiazovivin)

[0199] The The

[0199] reaction flaskflask reaction containing containing 2,4-dichloropyrimidine (372 mg, (372 2,4-dichloropyrimidine mg, 2.5 2.5 mmol), 6- mmol), 6 methoxy-1,2,3,4-tetrahydroquinoline (489 methoxy-1,2,3,4-tetrahydroquinoline (489 mg, mg, 33 mmol) and diisopropylethylamine mmol) and diisopropylethylamine (0.52 (0.52 mL, mL,

3 mmol) 3 mmol)in inn-butanol n-butanol (10(10 mL)mL) was heated was heated at 40 at 40 °C °C overnight. overnight. The was The solvent solvent was evaporated, evaporated,

and the and the residue residuewas purifiedby by waspurified flash flash column column chromatography to give to chromatography give 2-Chloro-4-(6 2-Chloro-4-(6-

25 mg, mg, (551(551 methoxy-3,4-dihydroquinolin-(2H)-yl)pyrimidine 25 methoxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine 80%). This This 80%). intermediate (250 (250 intermediate mg, 0.91 mg, 0.91 mmol) wasthen mmol)was dissolvedinin dichloromethane thendissolved andtreated dichloromethane and with BBr treated with (1 MMinin BBr 3(1

(1 mL, dichloromethane) (1 dichloromethane) -78 °C. mmol)atat -78 mL, 1I mmol) °C. The mixture was reaction mixture The reaction slowly warmed was slowly uptoto warmed up

temperature roomtemperature room and and stirred for for stirred I hr, 1 hr, poured poured into into water, water, extracted with with extracted dichloromethane. dichloromethane.

The combined The combinedorganics dried over weredried organicswere over anhydrous Na2SO anhydrousNaSO and4 and concentrated. The The concentrated. residue residue

30 waswas 30 by by purified purified flash column flashcolumn chromatography chromatography to give to give 2-Chloro-4-(6-hydroxy-3,4 2-Chloro-4-(6-hydroxy-3,4-

dihydroquinolin-1(2H)-yl)pyrimidine mg, To65%). (15465%). dihydroquinolin-1(2H)-yl)pyrimidine (154 mg, a stirred stirred solution To asolution of 2-chloro-4-(6 of 2-chloro-4-(6-

mg, 0.11 (29 mg, hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (29 hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine mmol)and 0.11 mmol) 4-amino-N and4-amino-N-

53

(27mg, (cyclopropylmethyl)benzenesulfonamide(27 (cyclopropylmethyl)benzenesulfonamide 0.12mmol) mg,0.12 mmol) DMF ininDMF (0.5 waswas mL)mL) (0.5 added p- p added acid(2(2M M toluenesulfonicacid toluenesulfonic in in dioxane) µL, pL, (55 (55 dioxane) 0.11 0.11 mmol). mmol). The reaction The reaction mixture mixture was was stirred stirred 90 °C at 90 at overnight,then °C overnight, purifiedbybyHPLC thenpurified HPLC to give to give the title the title compound (27 mg,(27 compound mg, 56%). 56%).

IZ S OH N N H N >N~ N OH IZ 2022204080 N H N N H 5 5

Example3:3:Reprogramming Example Reprogramming of human of human primary somaticsomatic primary cells cells by OCT4 OCT4 byand and chemical chemical

compounds compounds Herewewe

[02001 Here

[0200] reporta anovel report smallmolecule novelsmall cocktail that moleculecocktail enablesreprogramming that enables of reprogramming of

humanprimary human cells to somaticcells primarysomatic with exogenous iPSCs with to iPSCs exogenous expression of only expression of only OCT4. OCT4.

10 [0201] 10 [0201] severalseveral AmongAmong readily readily available available primary primary human human somatic cell cell somatic types, types, keratinocytes keratinocytes

that that can be easily can be easily isolated human fromhuman isolated from skinskin or hair or hair follicle follicle represent represent an attractive cellcell an attractive source source

for reprogramming, for reprogramming, because they endogenously because they endogenously express KLF4and express KLF4 andand cMYC, andcMYC, were were reported reported

to be to reprogrammed be reprogrammed moremore efficiently usingusing efficiently the conventional the conventional four four TFs threeor or TFs three TFs TFs (without (without MYC) MYC) (Aasen, et al., T. etT.al., (Aasen, NatNat Biotechnol Biotechnol 26:1276-1284 (2008); (2008); 26:1276-1284 N. et al.,N. Maherali,Maherali, Cell StemCell Stem et al., 15 3, 3,340-345(2008)). Cell 15 Cell More 340-345(2008)).More recently,wewe recently, reported thatdual reportedthat ofTGFP inhibition of dual inhibition and TGFß and

MAPK/ERK MAPK/ERK pathways usingusing pathways small small molecules molecules (i.e. (i.e. SB431542 and and SB431542 PD0325901, PD0325901, respectively) respectively)

providesaadrastically provides enhanced drastically enhanced condition for for condition reprogramming reprogramming offibroblasts of human with fourwith human fibroblasts four exogenousTFsTFs exogenous (i.e.OSKM) (i.e. OSKM) T. et T. (Lin, (Lin, al., al., Nat et Nat Methods Methods 6:805-808 (2009)). (2009)). 6:805-808 Furthermore, Furthermore, we we have shown have shownthat dual pathway such dual that such pathway inhibition couldalso inhibition could alsoenhance of human reprogramming of enhancereprogramming human

by by keratinocytes 20 keratinocytes 20 two two TFsTFs exogenous exogenous (i.e.OK) (i.e. OK) with with two two small molecules,Parnate smallmolecules, (an Parnate(an inhibitor of inhibitor lysine-specific demethylase of lysine-specific demethylase 1) and 1) and CHIR99021 CHIR99021 (a GSK3(ainhibitor) GSK3 inhibitor) (Li, (Li, W. et W. al., et al., Stem Cells 27:2992-3000 Stem Cells (2009)). However, 27:2992-3000 (2009)). 2-TFsreprogramming such2-TFs However,such reprogrammingprocess waswas process very very

inefficient and inefficient complex(e.g. and complex twotwo involving (e.g.involving exogenous exogenous TFs TFs and and four four chemicals), chemicals), and and reprogramming witheven reprogrammingwith oneless evenone TF appeared less TF TowardthetheOCT4 daunting. Toward appeared daunting. OCT4 only only

25 reprogramming, 25 reprogramming, we developed we developed a step-wise a step-wise strategy in in strategy refining reprogramming refiningreprogramming condition andand condition identifying reprogramming newreprogramming identifying new chemical chemical entities. entities. We first first attempted Weattempted to further further optimize to optimize the the reprogramming reprogramming process underunder process the four four the or or three three TFs (i.e. OSKM (i.e. or TFs OSKM OSK) condition condition or OSK) in in neonatal human neonatalhuman epidermal epidermal keratinocytes keratinocytes (NHEKs) (NHEKs) byvarious by testing various inhibitors testinginhibitors of TGFp of TGFß and and MAPK MAPK at at pathways pathways differentconcentrations different using previously concentrations using reported human previously reported iPSC human iPSC

30 methods characterizationmethods 30 characterization (Lin, al., Nat (Lin,T.T.etet al., Methods 6:805-808 NatMethods 6:805-808 (2009)). Encouragingly, we (2009)). Encouragingly, we

found that found combination of thecombination that the 0.5 M of0.5 µM PD0325901 PD0325901 and A-83-01(a (amore 0.5 µM MA-83-01 and0.5 more potent and potentand selective TGFp selective TGFß receptor receptor inhibitor) moremore waswas inhibitor) effective effective in enhancing in enhancing reprogramming reprogramming of human of human 54

keratinocytes transduced keratinocytes transduced with withOSKM OSK OSKM ororOSK (Figure8a). (Figure 8a).Remarkably, Remarkably,when we we when further further

reducedviral reduced transductionstotoonly viraltransductions only twotwo factors/OK, factors/OK, we could we could still still generate generate iPSCs iPSCs from from NHEKs NHEKs when when they they were were treatedwith treated with0.5 0.5µMpM and and PD0325901 PD0325901 0.5 0.5 pM A-83-01, µM A-83-01, although although with with

efficiency. Then low efficiency. low Then we we began began screening screening additional additional small molecules small molecules from a collection from a collection of of 5 known 5 known bioactive bioactive compounds compounds at various at various concentrations as as concentrations previouslyreported. previously reported. Among Among dozensofofcompounds dozens compounds tested tested so far, so far, surprisingly surprisingly we found we found that athat a small small molecule molecule activator activator of of 2022204080

PDK1 PDK1 (3'-phosphoinositide-dependent (3'-phosphoinositide-dependent kinase-1), kinase-1), PS48 (5 PS48 (5 has µM) that M) never that has beennever been reported reported in reprogramming, in reprogramming, cancan significantly significantly enhance enhance the reprogramming the reprogramming efficiency efficiency aboutfold. about fifteen fifteen fold. Interestingly, we Interestingly, wealso alsofound foundthat 0.25mMmM that0.25 sodium sodium butyrate butyrate (NaB, a(NaB, a histone histone deacetylase deacetylase

10 inhibitor) 10 inhibitor) turned turned outbetomuch out to be much more reliable more reliable and efficient and efficient than than the the previously previously reported reported 0.5 0.5 mMVPA mM VPA for for thethe generationofofiPSCs generation iPSCsunder underOKOK condition(Figure condition 8b). Subsequent (Figure8b). Subsequentfollow- follow up studies up demonstrated that studies demonstrated thatcombination combination of of5 5 iM µM PS48 and 0.25 PS48 and 0.25 mM NaB mM NaB couldfurther could further enhancethe enhance thereprogramming reprogramming efficiency efficiency over twenty-five over twenty-five fold (Figure fold (Figure 8b and Table 4).Table 8b and With 4). With such unprecedented such efficiency ininreprogramming unprecedented efficiency NHEKs reprogramming NHEKs under under only only two two TFs,wewe TFs, further further

15 explored 15 explored thethe of generating possibility of possibility generating iPSCs with OCT4 iPSCs with alonebybyrefining OCT4 alone refining combinations combinations of of those small those small molecules molecules during during different differenttreatment windows. treatment windows.Primary Primary NHEKs NHEKs were transduced weretransduced with OCT4 with andtreated OCT4 and with chemicals treated with chemicals (Figure (Figure 8c). 8c). Among variousconditions, Among various conditions, small small iPSC iPSC

colonies resembling colonies resembling hESCs hESCs (four(four six colonies to colonies to six out1,000,000 out of of 1,000,000 seeded seeded cells) appeared cells) appeared in in OCT4infected OCT4 infectedNHEKs NHEKs that that were were treatedwith treated with0.25 0.25 mM mM NaB, NaB, 5 PS48 5 µM M PS48 and µM and 0.5 0.5 A-83- pM A-83 20 01 during 20 01 during thethe firstfour first weeks, followed four weeks, followed by by treatment treatment with 0.25 mM with 0.25 NaB,5 5µMpM mM NaB, PS48, PS48, 0.50.5

gMA-83-01 µM A-83-01and and0.5 0.5µMpM PD0325901 PD0325901 for another for another fourfour weeks weeks (Figure (Figure 8c). 8c). Such Such TRA-1-81 TRA-1-81

positive iPSC positive iPSCcolonies (Figure colonies(Figure 8d) 8d) grew grew larger larger underunder conventional conventional hESC media hESC culture culture andmedia and could bebeserially could passagedto toyield serially passaged yieldstable stableiPSC iPSC thatthat clones clones werewere further further characterized characterized (Figure (Figure

8e and 8e and9). 9). More More significantly, significantly, OCT4 OCT4 only only iPSCs iPSCs could could also be also be generated generated from from human human adult adult keratinocytes 25 keratinocytes 25 by by additionofof22µM MParnate addition and3 3µM M Parnateand CHIR99021 CHIR99021 (which (which had been shownshown had been to to improve reprogramming improve reprogrammingofofNHEKs NHEKs under under OK condition) OK condition) to this to this chemical chemical cocktail.After cocktail. Afterthe the reliable reprogramming reliable of primary reprogramming of keratinocytestotoiPSCs primary keratinocytes iPSCsby byOCT4 and small OCT4 and molecules, we small molecules, we

applied the further applied further the conditions conditionstotoother otherhuman human primary primary cell types, cell types, including HUVECsHUVECs including (differentiated mesoderm (differentiated mesoderm cells) cells) andand AFDCs AFDCs (amniotic (amniotic fluid derived fluid derived cells). Similarly, cells). Similarly, TRA-1- TRA-1 30 81 positive 30 81 positive iPSC iPSC coloniesappeared colonies appearedin inOCT4 OCT4 infected infected HUVECs HUVECs and AFDCs and AFDCs thattreated that were were treated with chemicals. with Remarkably, itit appeared chemicals. Remarkably, that reprogramming appeared that reprogramming of HUVECs of HUVECs andand AFDCs AFDCs was was more efficient more efficient and andfaster fasterthan reprogramming than reprogrammingof ofNHEKs under the NHEKs under the OCT4 OCT4and andsmall smallmolecule molecule (Table4).4).TwoTwo conditions(Table conditions clones clones of iPSCs of iPSCs fromcell from each cellwere eachtype type were long-term long-term expanded expanded for for over 20passages over 20 passagesunder under conventional conventional cultureculture hESC hESC condition condition and characterized and further further characterized (Table (Table 5). 35 5). 35 55

[0202] These

[0202] These stablyexpanded stably expandedhiPSC-OK hiPSC-OK and and hiPSC-O hiPSC-O cellscells are are morphologically morphologically

indistinguishable tohESCs, indistinguishable to hESCs,andand could could be cultured be cultured on ECM-coated on ECM-coated surface surface under under feeder-free feeder-free

and chemically and chemicallydefined defined conditions conditions (Figure (Figure 8e Figure 8e and and Figure 13). stained 13). They They stained positive positive for for alkaline phosphatase alkaline phosphatase(ALP) (ALP) and and expressed expressed typical typical pluripotency pluripotency markers, markers, including OCT4, including OCT4, 55 SOX2, SOX2, NANOG, NANOG, TRA-1-81 TRA-1-81 anddetected and SSEA4, SSEA4, by detected by immunocytochemistry/ICC immunocytochemistry/ICC (Figure 8e,(Figure 8e, 10b, Figures 10b, Figures11-12). 11-12).In Inaddition, addition,RT-PCR RT-PCR analysis analysis confirmed confirmed the expression the expression of the of the 2022204080

endogenous human endogenous human OCT4, OCT4, SOX2, SOX2, NANOG, REX],UTF1, NANOG, REX1, UTF,TDGF2, TDGF2,FGF4 FGF4 genes,and genes, and silencing of silencing ofexogenous exogenous OCT4 OCT4 and(Figure and KLF4 KLF4 (Figure 9a and 9a and 10c). 10c). Furthermore, Furthermore, bisulfite bisulfite sequencing analysis sequencing analysis revealed revealed that thatthe OCT4 the OCT4and andNANOG promotersofofhiPSC-OK NANOG promoters hiPSC-OKand and

10 hiPSC-O 10 hiPSC-O cellslargely cells are are largely demethylated demethylated (Figure (Figure 9b 9b and and 10d). ThisIOd). resultThis resultfurther provides provides further evidence forreactivation evidence for reactivationofofthe thepluripotency pluripotency transcription transcription program program in hiPSC-OK in the the hiPSC-OK and and hiPSC-Ocells. hiPSC-O cells. Global Global gene gene expression expression analysis analysis of ofhiPSC-O cells, NHEKs hiPSC-O cells, and hESCs NHEKs and hESCs showedthat showed thathiPSC-O hiPSC-O cells cells are are distinct distinct fromfrom NHEKsNHEKs (Pearson(Pearson correlation correlation value: value: 0.87) and 0.87) and mostsimilar most similartotohESCs hESCs (Pearson (Pearson correlation correlation value: value: 0.98)0.98) (Figure (Figure 9c). Genetyping 9c). Genetyping analysis analysis 15 showed 15 showed thatthat hiPSC-O hiPSC-O cells cells onlyonly contained contained thethe OCT4 OCT4 transgene transgene without without thethe contamination contamination of of

transgeneKLF4 transgene KLF4 or SOX2 or SOX2 (Figure (Figure 15). Southern 15). Southern blot analysis blot analysis showed showed that therethat werethere were multiple multiple different integration different integration sites sites of of the OCT4 the OCT4 transgene transgene (Figure (Figure 16) among 16) among different different clones. clones. In In addition, karyotyping addition, karyotypingresult resultdemonstrated demonstrated thatthat hiPSC-O hiPSC-O maintained maintained normal karyotype normal karyotype during during the whole the reprogrammingand whole reprogramming andexpansion expansionprocess process(Figure (Figure 17). 17). Furthermore, Furthermore, DNA DNA 20 fingerprinting 20 fingerprinting test test excluded excluded the possibility the possibility that these that these hiPSCshiPSCs arose arose from from hESC hESC contamination contamination

in the in the laboratory (Table6). laboratory (Table 6). ToTo examine examine the developmental the developmental potential potential ofhiPSC-O of these cells, these hiPSC-O cells, they were they weredifferentiated differentiatedininvitro vitrobybythe thestandard standard embryoid embryoid body body (EB) differentiation (EB) differentiation method.method.

ICCanalyses ICC analysesdemonstrated demonstrated that that they they couldcould effectively effectively differentiate differentiate into pII-tubulin* into BIII-tubulin

characteristic neuronal characteristic neuronalcells (ectoderm), cells SMA* (ectoderm), SMA mesodermal cells, and mesodermal cells, andAFP* endodermalcells AFP endodermal cells 25 (Figure 25 (Figure 9d 9d andand IOe). 10e). QuantitativePCRPCR Quantitative analyses analyses furtherconfirmed further confirmedthe theexpression expressionofofthese these and additional and additionallineage lineagespecific specificmarker marker genes, genes, including including ectodermal ectodermal cells (#1I-tubulin cells (III-tubulin and and NESTIN), mesodermal NESTIN), mesodermalcells cells(MSXI (MSX]andand MLC2a), MLC2a), and and endodermal endodermal cellscells (FOXA2 (FOXA2 and AFP) and AFP)

(Figure 9e). (Figure 9e). Following FollowingEB EB protocol, protocol, thesethese hiPSC-OK hiPSC-OK and cells and hiPSC-O hiPSC-O could cells also could also give rise give rise to to rhythmically beatingcardiomyocytes. rhythmically beating cardiomyocytes. Totheir To test test their in vivo in vivo pluripotency, pluripotency, they were they were

30 transplanted 30 transplanted into into SCID SCID mice. Four-six mice. Four-six weeks weeks later, later, these thesecells hiPSC-O hiPSC-O cells effectively effectively generated generated typical teratomascontaining typical teratomas containing derivatives derivatives of all of all three three germ germ layers layers (Figure (Figure 9f 10f). 9f and and IOf). Collectively, theseininvitro Collectively, these vitroand andininvivo vivocharacterizations characterizationsdemonstrated demonstrated that that a single a single

transcription factor, transcription factor, OCT4, OCT4, combined combined with with a defined a defined small small molecule molecule cocktailcocktail is sufficient is sufficient to to reprogram several reprogram several human human primary primary somatic somatic cells cells to to that iPSCs iPSCsarethat are morphologically, morphologically,

35 molecularly 35 molecularly andand functionallysimilar functionally similartoto pluripotent pluripotent hESCs. hESCs.

56

[0203] The

[0203] The studiespresented studies presentedabove abovehave havea anumber numberofofimportant importantimplications: implications: (1) (1) Although Although

fetal NSCs fetal were NSCs were shown shown toreprogrammed to be be reprogrammed to iPSCstobyiPSCs byexpression ectopic ectopic expression of Oct4 of Oct4 alone, alone, there havebeen there have beensignificant significantskepticisms skepticisms around around whether whether exogenous exogenous Oct4 Oct4 gene gene alone alone would be would be

sufficient to sufficient to reprogram othermore reprogram other more practical practical human human somatic somatic cells dothat cells that notdo not endogenously endogenously

55 express express Sox2 Sox2 (one (one of theof themaster two two master pluripotency pluripotency genes in genes in reprogramming), reprogramming), are at later are at later developmental developmental stages stages (e.g. (e.g. early early embryonic/fetal embryonic/fetal vs. born/adult), VS. born/adult), andbecan and can be obtained obtained withoutwithout 2022204080

significant harms significant harmstotothe individual.To To theindividual. ourour knowledge, knowledge, our study is theisfirst our study the first demonstration demonstration

that iPSCs that canbebepractically iPSCs can practicallyderived derived from from readily readily available available primary primary human human somatic somatic cells cells (e.g. keratinocytes) (e.g. transducedwith keratinocytes) transduced with a single a single exogenous exogenous reprogramming reprogramming gene, gene, Oct4. In Oct4. In 10 contrast 10 contrast to neural to neural stem stem cellscells from from the brain, the brain, keratinocytes keratinocytes areaccessible are more more accessible and and can be can be easily obtained easily obtainedfrom fromborn born individuals individuals withwith lessless invasive invasive procedures. procedures. This further This further strengthens strengthens

the strategy of the strategy of exploiting exploitingvarious variouspractically practicallyaccessible accessiblehuman human somatic somatic cells cells for iPSC for iPSC

generationwith generation withsafer saferapproaches approaches and/or and/or better better qualities. qualities. Thus, Thus, this this new method new method and its and its further development further development would would significantly significantly facilitate facilitate production production of patient-specific of patient-specific pluripotent pluripotent

15 stemstem 15 cellscells for various for various applications. applications. (2) Although (2) Although small molecules and their and small molecules their combinations combinations

havebeen have beenidentified identifiedtotoreplace replaceonly only oneone or or twotwo reprogramming reprogramming TFs, it TFs, it becomes becomes exponentially exponentially

challenging to challenging generateiPSCs to generate iPSCs when when more reprogramming exogenousreprogramming more exogenous TFsTFs areare omitted omitted

together. Theidentification together. The identificationofof thisnew this new small small molecule molecule cocktail, cocktail, whichwhich functionally functionally re-places re-places

three transcriptionfactors mastertranscription three master factorsall alltogether (i.e. Sox2, together(i.e. Sox2,Klf4 Klf4andand cMyc) cMyc) in enabling in enabling

20 generation 20 generation of iPSCs of iPSCs withalone, with Oct4 Oct4 represents alone, represents another another major stepmajor towardstep the toward ultimatethe ultimate reprogramming reprogramming withwith only only smallsmall molecules, molecules, and further and further proved proved and and solidified solidified the the chemical chemical approachtotoiPSCs. approach iPSCs. (3) (3) This This demonstrated demonstrated singlesingle gene condition gene condition also hasalso has a significant a significant

implicationfor implication forprotein-induced protein-induced pluripotent pluripotent stemstem cell cell (piPSC) (piPSC) technology. technology. A practical A practical

challengefor challenge forpiPSC piPSC technology technology is large-scale is large-scale and reliable and reliable production of the of production thetransducible four four transducible 25 reprogramming 25 reprogramming proteins, proteins, each of each whichof whichdifferently behaves behaves differently in manufacture in manufacture (e.g. their (e.g. their expression,folding, expression, folding,stability stability etc.). etc.). Clearly, Clearly, combining combining this this small small molecule molecule cocktail cocktail with with a a single transducible single transducibleprotein proteinwould would significantly significantly simplify simplify the the piPSC piPSC technology technology and facilitate and facilitate

its applications. its (4) More applications. (4) significantly,wewe Moresignificantly, identified identified a new a new small small molecule, molecule, PS48, PS48, with a with a new target/mechanism new target/mechanism inin enhancing enhancing reprogramming. reprogramming.PS48 PS48 is an is an allosteric small allosteric small molecule molecule 30 activator 30 activatorofofPDK1, PDKI, which which is is anan importantupstream important upstream kinasefor kinase forseveral several AGC AGCkinases, kinases,including including Akt/PKB Akt/PKB (Alessi (Alessi et al.,Curr et al., Curr Biol Biol 7, 261-269 7, 261-269 (1997)). (1997)). Its reprogramming Its reprogramming enhancing enhancing effect effect maybebepartly may attributedtotothe partlyattributed theactivation activationofof Akt/PKB, Akt/PKB, whichwhich promotes promotes cell proliferation cell proliferation and and survival (Manning, survival (Manning,B. B. D.,D., Cantley, Cantley, L. C., L. C., CellCell 129,129, 1261-1274 1261-1274 (2007)). (2007)). Further Further in-depth in-depth

characterizations ononhow characterizations how PDKI-involved mechanisms PDK1-involved mechanisms areare preciselyregulated precisely regulated during during 35 reprogramming 35 reprogramming process process should should provide provide additional additional insightsunderlying insights underlyingreprogramming reprogramming andand

57

pluripotency. Furthermore, pluripotency. Furthermore, because because mightmight therethere begreater be even even greater hidden(e.g. hidden risks (e.g. risksmore more subtle genetic subtle genetic and/or epigeneticabnormalities and/orepigenetic abnormalities could could be generated be generated or selected or selected duringduring the the reprogramming reprogramming process) process) imposed imposed by theby lowthe and slowand low efficiency efficiency slow kinetics kinetics of reprogramming, of reprogramming,

identification of identification of new smallmolecules new small molecules for for enhancing enhancing reprogramming reprogramming as illustrated again inagain in as illustrated 5 thisthis 5 study study would would always always be highly be highly valuable valuable safer, a toward atoward safer,and easier easier more and more efficient efficient forhuman procedurefor procedure humaniPSCiPSC generation. generation. (5) Finally, (5) Finally, thisand this new new and powerful powerful small small molecule molecule 2022204080

cocktail for cocktail for reprogramming reprogramming validated validated the the step-wise step-wise chemical chemical optimization optimization and screening and screening

strategy presented strategy presentedhere productive hereasasa aproductive approach approach toward toward the ultimate purelypurely the ultimate chemical chemical-

inducedpluripotent induced pluripotentstem stem Moreover, cells.Moreover, cells. the finding the finding that that different smallsmall different molecules molecules

10 modulating 10 modulating the target/mechanism the same same target/mechanism could havecould have significantly significantly different different effects on effects on a different reprogramming in aindifferent reprogramming context, context, exemplified exemplified by A-83-0 by A-83-01's and l's NaB's NaB's better andbetter reprogramming reprogramming enhancing enhancing activities activities in human in human keratinocytes, suggestssuggests keratinocytes, the importance the importance of of optimization "individualized"optimization "individualized" andand treatment treatment withwith different different regimens regimens for specific for specific

reprogrammingcontext. reprogramming context.

15 CellCulture 15 Cell Culture

[0204] Normal

[0204] Normal Human Human Epidermal Epidermal Keratinocytes Keratinocytes (Lonza) (Lonza) were were maintained maintained in Keratinocyte in Keratinocyte

culturing culturing medium (KCM,Lonza). medium (KCM, Lonza).Human Human Umbilical Umbilical VeinVein Endothelial Endothelial Cells Cells (HUVECs, (HUVECs,

Millipore)were Millipore) weremaintained maintained CompleteMedium in EndoGRO-VEGFComplete in EndoGRO-VEGF Medium(HCM, (HCM, CHEMICON). CHEMICON).

Human ESCs Human ESCs andand hiPSCs hiPSCs were were cultured cultured on on MEFMEF feeder feeder cells cells in in conventionalhuman conventional ESCESC human 20 culture 20 culturemedia media (hESCM: (hESCM:DMEM/F12, DMEM/F12,15%15% Knockout Knockout serum serum replacement, 1%Glutamax, replacement, 1% Glutamax, 1% 1%

amino Non-essentialamino Non-essential acids, acids, 1% penicillin/streptomycin, 0.1 mM0.1 1% penicillin/streptomycin, mM3-mercaptoethanol ß-mercaptoethanol and 10 and 10 ng/ml bFGF). All ng/ml bFGF). Allcell culture products cell culture productswere were from Invitrogen/Gibco BRL from Invitrogen/Gibco except where BRL except where mentioned. mentioned.

Lentivirus Production Lentivirus Production

25 [0205] 25 [0205] The lentivirus The lentivirus supernatants supernatants werewere produced and and produced harvested harvested as previously as previously described described

(Yu, J. (Yu, J. et al.,Science et al., Science 318:1917-1920 (2007)). 318:1917-1920 (2007)). The The plasmids plasmids used used for for lentivirus lentivirus production production

include pSin-EF2-Puro-hOCT4, include pSin2-EF2-Puro-hSOX2, pSin-EF2-Puro-hOCT4, pSin2-EF2-Puro-hSOX2, pLove-mKlf4, pLove-mKlf4, pLove-mMyc, pLove-mMyc, the the packaging plasmid packaging psPAX2 plasmid psPAX2 and and thethe envelop-codingplasmid envelop-coding pMD2.G plasmidpMD2.G (Yu, (Yu, J. al., J. et et al.,Science Science 318:1917-1920 318:1917-1920 (2007) (2007) Li,etW. and W. and Li, et Stem al., al., Stem Cells Cells 27:2992-3000 (2009)). (2009)). 27:2992-3000

30 Reprogramming 30 ReprogrammingofofNHEKs NHEKs

[02061 NHEKs

[0206] werewere NHEKs cultured cultured a 100 in ain100 mm mm tissue tissue culture culture dishand dish transduced3 3times andtransduced times(3-4 (3-4 hours each hours eachtransduction) with transduction)with freshly freshly produced produced lentivirus lentivirus supernatants. supernatants. 1,000,000 1,000,000 transduced transduced

58

NHEKs NHEKs were were seeded seeded on on thethe irradiated x-ray irradiated inactivated CF x-ray inactivated IMEF CF1 MEF feeder cells inina100-mm feeder cells a 100-mm

dish and dish culturedininKCM and cultured treated with and treated KCM and with 55 µM M PS48, 0.25 mM PS48, 0.25 NaB mM NaB (Stemgent) andand (Stemgent) 0.5 0.5

gMA-83-01 µM A-83-01(Stemgent) for22weeks, (Stemgent)for followedby weeks, followed by changing changinghalf volumeofofmedia half volume mediato to hESCM hESCM supplementing with and supplementing and with 55 µMMPS48, PS48,0.25 NaBNaB 0.25 mMmM and and 0.5 0.5 µM gM A-83-01 A-83-01 for another for another 2 weeks. 2 weeks.

5 Then 5 Then cellcell mediawere culturemedia culture werechanged to to changed hESCM hESCM and and supplemented supplemented 5 µM 5PS48, with with M PS48, 0.25 0.25 mMNaB, mM NaB, 0.5µMpM 0.5 A-83-01 and and A-83-01 0.5 0.5 M PD0325901 µM PD0325901 (Stemgent) (Stemgent) for additional for additional four four weeks. weeks. 2022204080

The sameOCT4 The same OCT4 infected infected keratinocytes keratinocytes cultured cultured in without in media chemicalschemicals media without as a used as a were used were

control. The control. culturewaswas Theculture splitbyby split Accutase Accutase (Millipore) (Millipore) and treated with 1with and treated I pM Thiazovivin µM Thiazovivin

(Stemgent)ininthe (Stemgent) thefirst first day after splitting. dayafter splitting. The iPSC TheiPSC colonies colonies stained stained positive positive by Alexa Fluor Fluor by Alexa 10 555555 10 Mouse Mouse anti-Human anti-Human TRA-1-81 TRA-1-81 antibody antibody (BD Pharmingen) (BD Pharmingen) wereup were picked picked up for expansion for expansion

feeder cells on feeder on cells in in hESCM hESCM and and cultured cultured routinely. routinely.

Reprogramming of Reprogramming ofHUVECs HUVECs

HUVECs

[02071 HUVECs

[0207] were were cultured cultured in a in100 a 100 mm tissue mm tissue culture culture dish dish and and transduced2 2times transduced times(4-6 (4-6 hours each hours transduction)with eachtransduction) with freshly freshly produced produced lentivirus lentivirus supernatants. 200,000200,000 supernatants. transduced transduced

15 HUVECs 15 HUVECs were seeded were seeded on gelatin on gelatin coated coated 100-mm 100-mm dish, dish, cultured cultured in HCM, in HCM, and treated and treated with with 5 5 gMPS48, µM PS48,0.25 mM 0.25mM NaBNaB and and 0.5 0.5 for 2for M A-83-01 µM A-83-01 2 weeks, weeks, followed followed by changing by changing halfhalf

volume of volume of media media to to hESCM hESCM andand supplementing supplementing with with 5 PS48, 5 µM 0.250.25 M PS48, mMand mM NaB NaB0.5 andµM0.5 gM for another A-83-01 for A-83-01 another 22 weeks. cell culture Then cell weeks. Then culturemedia media were were changed changed to to hESCM and hESCM and

with 55 µM supplemented with supplemented pMPS48, PS48,0.25 0.25mMmM NaB, NaB, 0.5 0.5 µM pM A-83-01 A-83-01 and µM and 0.5 0.5PD0325901 M PD0325901 for for 20 additional 20 additional1-21-2weeks. weeks.TheThe iPSC iPSC colonies colonies stained stained positivebybyAlexa positive AlexaFluor 555Mouse Fluor555 Mouse anti anti-

HumanTRA-1-81 Human TRA-1-81 antibody antibody were were picked picked up for up for expansion on on expansion feeder feeder cellsinin hESCM cells hESCM andand

routinely. The cultured routinely. cultured culture Theculture waswas split by by split Accutase Accutase and treated with 1with and treated I pM Thiazovivin µM Thiazovivin in in the first day after splitting. the first day after splitting.

In vitro In vitro Differentiation Differentiation

25 [0208] 25 [0208] The The in in vitro vitro differentiationofofhiPSCs differentiation hiPSCswaswas carriedout carried outbybythe the standard embryoid standard embryoid body (EB)method. body (EB) method. Briefly, Briefly, the hiPSCs the hiPSCs were dissociated were dissociated by Accutase by Accutase (Millipore), (Millipore), cultured in cultured in

ultra-low attachment6-well ultra-low attachment 6-well plate plate forfor eight eight days days and and thenthen transferred transferred to Matrigel-coated to Matrigel-coated 6- 6 well in differentiation plate in well plate differentiation medium. medium. The The cells cells fixedfixed werewere for immunocytochemical analysis analysis for immunocytochemical harvested for or harvested or forRT-PCR tests eight RT-PCR tests eightdays dayslater. Differentiation later. medium: Differentiation DMEM/F12, medium: 10% DMEM/F12, 10%

30 FBS, FBS, 1% Glutamax, 1% Glutamax, 1% Non-essential aminoamino 1% Non-essential acids,acids, 1% penicillin/streptomycin, 0.1 0.1 1% penicillin/streptomycin, mM mM ß-

mercaptoethanol. mercaptoethanol.

59

Alkaline Alkaline Phosphatase Stainingand PhosphataseStaining Assay Assay Immunocytochemistry andImmunocytochemistry

AlkalinePhosphatase

[0209] Alkaline

[0209] stainingwas Phosphatasestaining the manufacturer's accordingtotothe performedaccording wasperformed manufacturer's protocol theAlkaline usingthe protocol using Phosphatase AlkalinePhosphatase Detection Detection Kit (Stemgent). StandardStandard Kit (Stemgent). immunocytochemistry immunocytochemistry assayassay out as out was carried was carried previously reportedreported as previously (Li,al., (Li, W. et et al., W.Stem Stem Cells Cells 5 27:2992-3000 5 27:2992-3000 (2009)). (2009)). Primary Primary antibodies antibodies used used be be cancan found in in found theTable the Secondary Table3.3. Secondary

2022204080 wereAlexa antibodieswere antibodies Alexa Fluor Fluor 488 488 donkey donkey anti-mouse anti-mouse or anti-rabbit or anti-rabbit IgG (1:1000) IgG (1:1000) (Invitrogen). (Invitrogen).

Nuclei werevisualized Nuclei were by by visualized DAPI DAPI (Sigma-Aldrich) (Sigma-Aldrich) staining. staining. Images Images were captured captured wereusing a using a Nikon Eclipse TE2000-U Nikon Eclipse microscope. TE2000-Umicroscope.

Gene Expression Analysis Gene Expression and qRT-PCR RT-PCR and AnalysisbybyRT-PCR qRT-PCR

10 [0210] 10 [0210] For RT-PCR For RT-PCR and qRT-PCR analysis,analysis, and qRT-PCR total RNAtotal wasRNA was extracted extracted fromiPSCs from human human iPSCs using the using RNeasy the RNeasy Plus Plus Mini Mini Kit in in combination Kitcombination with QAshredder with QIAshredder (Qiagen). (Qiagen). First First strand strand reverse transcription reverse transcription wasperformed was performedwith with2 2 gµgRNA RNA using using iScriptTM iScript cDNA cDNASynthesis Kit Synthesis Kit

Theexpression (BioRad). The (BioRad). expression of markers was pluripotency markers of pluripotency was analyzed by RT-PCR analyzed by usingPlatinum RT-PCRusing Platinum SuperMix PCRSuperMix PCR (Invitrogen). (Invitrogen). The expression The expression of lineage specificspecific of lineage markers markers after after differentiation differentiation

15 waswas 15 analyzed analyzed by qRT-PCR usingusing by qRT-PCR iQ SYBR iQ SYBR Green Supermix Green Supermix (Bio-Rad). (Bio-Rad). The primers primers The can be can be found inthe found in Table2.2. the Table

Analysis MicroarrayAnalysis Microarray

The

[0211] The

[0211] Human Human Ref-8_v3 Ref-8_v3 expression expression Beadchip Beadchip (Illumina, USA)USA) CA, CA, (Illumina, was used used was for for microarray hybridizations microarray examine the hybridizations totoexamine theglobal geneexpression globalgene expression NHEKs, ofof hiPSC and NHEKs, hiPSC and hES hES

20 cells. 20 Biotin-I6-UTP-labeled cells.Biotin-16-UTP-labeled cRNA cRNA was synthesized from from was synthesized 500 ng ng total 500total with with RNA RNA the the TotalPrep RNA Illumina TotalPrep Illumina kit (Ambion amplification kit RNA amplification AMIL1791, (Ambion AMIL1791, Foster Foster City,CA, City, TheThe USA). CA,USA). hybridization 750 750 containing mixcontaining hybridizationmix of labeled nglabeled ng of amplified amplified cRNA cRNA was was according prepared according prepared to to the Illumina BeadStation the Illumina 500x System BeadStation 500x Manual(Illumina, System Manual CA,USA) Diego, CA, San Diego, (Illumina, San USA)using the usingthe supplied andGE GE reagentsand supplied reagents Healthcare Healthcare Streptavidin-Cy3 stainingstaining Streptavidin-Cy3 solution. solution. Hybridization Hybridization to to 25 thethe 25 Illumina Illumina Human Human Ref-8_v3 Ref-8_v3 expression expression Beadchip Beadchip was for h ath 55 forl8 was 18 at 55 on on °C °C a BeadChip Hyb Hyb a BeadChip Wheel. Thearray Wheel. The wasscanned array was usingthe scannedusing Illumina BeadArray the Illumina BeadArray Reader. Allsamples Reader. All were sampleswere preparedinintwo prepared biologicalreplicates. twobiological Processing replicates.Processing and analysis and analysis of the the microarray ofmicroarray data data were were performedwith performed thethe with Illumina Illumina BeadStudio BeadStudio software. software. The The data data were were subtracted subtracted for background for background

normalized and normalized and using thethe using rank rank invariant invariant option. option.

30 Bisulfate 30 BisulfateGenomic Sequencing GenomicSequencing

[0212] Genomic

[0212] Genomic DNAs DNAs were were isolated isolated using the the using Non Non Organic Organic DNA Isolation DNA Isolation Kit (Millipore) Kit (Millipore)

treatedwith and then treated and withthe EZEZDNA the DNA Methylation-Gold Kit (Zymo Methylation-GoldKit (ZymoResearch Orange, Corp.,Orange, ResearchCorp.,

60

CA). TheThe CA). treated treated DNAs DNAs were then thenasused wereused as templates templates to amplify sequencessequences to amplify of interest. of interest.

Primers used Primers for OCT4 used for andNANOG OCT4 and NANOG promoter promoter fragment fragment amplification are are amplification indicatedin inTable indicated Table 2. The 2. The resulting were cloned fragments were resulting fragments cloned using theTOPO usingthe TACloning TOPO TA CloningKit sequencing for sequencing Kit for andsequenced. (Invitrogen) and (Invitrogen) sequenced.

55 Genotyping of of Genotyping hiPSCs hiPSCs

2022204080

[0213] of of Genotyping

[0213] Genotyping hiPSC hiPSC lineswas lines was performed performed using using RT-PCR RT-PCR of genomic of genomic DNA DNA with with specific specific primers Yu,J.J.etetal., (Table2;2;Yu, primers(Table al., Science 318:1917-1920 Science318:1917-1920 (2007) (2007) andW.Li, and Li, et W. al., Stem al.,etStem

Cells 27:2992-3000 Cells 27:2992-3000 (2009)). (2009)).

Formation Teratoma Formation Teratoma

10 [0214] 10 [0214] The hiPSC were were lines lines The hiPSC harvested harvested by using by using 0.05 0.05 % Trypsin-EDTA. % Trypsin-EDTA. Five million Five million cells cells were injectedunder were injected kidney underthethekidney capsule capsule of SCID of SCID mice (n=3). mice (n=3). After After 4-6 weeks, weeks, 4-6well well developed developed

teratomas teratomas were harvested, wereharvested, fixed and and fixed thenthen histologically histologically analyzed analyzed at TSRI TSRI histology at histology core core facility. facility.

Table 22 Primers Table Primersused used

Gene Gene Forward Forward Reverse Reverse

For RT-PCR For RT-PCR Endo-OCT4 Endo-OCT4 AGTTTGTGCCAGGGTTTTTG AGTTTGTGCCAGGGTTTTTG ACTTCACCTTCCCTCCAACC ACTTCACCTTCCCTCCAACC Endo-SOX2 Endo-SOX2 CAAAAATGGCCATGCAGGTT CAAAAATGGCCATGCAGGTT AGTTGGGATCGAACAAAAGCTATT AGTTGGGATCGAACAAAAGCTATT Endo- Endo- TTTGGAAGCTGCTGGGGAAG TTTGGAAGCTGCTGGGGAAG GATGGGAGGAGGGGAGAGGA GATGGGAGGAGGGGAGAGGA NANOG NANOG Endo-KLF4 Endo-KLF4 ACGATCGTGGCCCCGGAAAAGGACC ACGATCGTGGCCCCGGAAAAGGACC GATTGTAGTGCTTTCTGGCTGGGCTCC GATTGTAGTGCTTTCTGGCTGGGCTCC Endo-cMYC Endo-cMYC GCGTCCTGGGAAGGGAGATCCGGAGC GCGTCCTGGGAAGGGAGATCCGGAGC TTGAGGGGCATCGTCGCGGGAGGCTG TTGAGGGGCATCGTCGCGGGAGGCTG REX] REXI CAGATCCTAAACAGCTCGCAGAAT CAGATCCTAAACAGCTCGCAGAAT GCGTACGCAAATTAAAGTCCAGA GCGTACGCAAATTAAAGTCCAGA UTFI UTFI CCGTCGCTGAACACCGCCCTGCTG CCGTCGCTGAACACCGCCCTGCTG CGCGCTGCCCAGAATGAAGCCCAC CGCGCTGCCCAGAATGAAGCCCAC TDGF2 TDGF2 CTGCTGCCTGAATGGGGGAACCTGC CTGCTGCCTGAATGGGGGAACCTGC GCCACGAGGTGCTCATCCATCACAAGG GCCACGAGGTGCTCATCCATCACAAGG FGF4 FGF4 CTACAACGCCTACGAGTCCTACA CTACAACGCCTACGAGTCCTACA GTTGCACCAGAAAAGTCAGAGTTG GTTGCACCAGAAAAGTCAGAGTTG Exo-OCT4 Exo-OCT4 TGTCTCCGTCACCACTCTGG TGTCTCCGTCACCACTCTGG ATGCATGCGGATCCTTCG ATGCATGCGGATCCTTCG PAX6 PAX6 TGTCCAACGGATGTGAGT TGTCCAACGGATGTGAGT TTTCCCAAGCAAAGATGGAC TTTCCCAAGCAAAGATGGAC fII BIII CAACAGCACGGCCATCCAGG CAACAGCACGGCCATCCAGG CTTGGGGCCCTGGGCCTCCGA CTTGGGGCCCTGGGCCTCCGA TUBULIN TUBULIN FOXF1 FOXFI AAAGGAGCCACGAAGCAAGC AAAGGAGCCACGAAGCAAGC AGGCTGAAGCGAAGGAAGAGG AGGCTGAAGCGAAGGAAGAGG HANDI TCCCTTTTCCGCTTGCTCTC TCCCTTTTCCGCTTGCTCTC CATCGCCTACCTGATGGACG CATCGCCTACCTGATGGACG HANDI AFP AGCAGCTTGGTGGTGGATGA AGCAGCTTGGTGGTGGATGA CCTGAGCTTGGCACAGATCCT CCTGAGCTTGGCACAGATCCT AFP GA TA 6 GATA6 TGTGCGTTCATGGAGAAGATCA TGTGCGTTCATGGAGAAGATCA TTTGATAAGAGACCTCATGAACCGACT TTTGATAAGAGACCTCATGAACCGACT GAPDH GTGGACCTGACCTGCCGTCT GTGGACCTGACCTGCCGTCT GGAGGAGTGGGTGTCGCTGT GGAGGAGTGGGTGTCGCTGT GAPDH Forbisulfate-sequencing For bisulfate-sequencing OCT4-1 OCT4-1 TTAGGAAAATGGGTAGTAGGGATTT TTAGGAAAATGGGTAGTAGGGATTT TACCCAAAAAACAAATAAATTATAAAACCT TACCCAAAAAACAAATAAATTATAAAACCT OCT4-2 OCT4-2 GGATGTTATTAAGATGAAGATAGTTGG GGATGTTATTAAGATGAAGATAGTTGG CCTAAACTCCCCTTCAAAATCTATT CCTAAACTCCCCTTCAAAATCTATT NANOG GAGTTAAAGAGTTTTGTTTTTAAAAATTAT GAGTTAAAGAGTTTTGTTTTTAAAAATTAT TCCCAAATCTAATAATTTATCATATCTTTC TCCCAAATCTAATAATTTATCATATCTTTC NANOG 61

Gene Gene Forward Forward Reverse Reverse

For genotyping For genotyping

OCT4-Int OCT4-Int CAGTGCCCGAAACCCACAC CAGTGCCCGAAACCCACAC AGAGGAACTGCTTCCTTCACGACA AGAGGAACTGCTTCCTTCACGACA SOX2-Int SOX2-Int TACCTCTTCCTCCCACTCCA TACCTCTTCCTCCCACTCCA AGAGGAACTGCTTCCTTCACGACA AGAGGAACTGCTTCCTTCACGACA KLF4-Int KLF4-Int CACCTTGCCTTACACATGAAGAGG CACCTTGCCTTACACATGAAGAGG CGTAGAATCGAGACCGAGGAGA CGTAGAATCGAGACCGAGGAGA

Table 33 Primary Table Primaryantibodies applied antibodiesapplied 2022204080

Antibody Antibody Species Species Dilution Dilution Vendor Vendor (1) Anti-OCT4(1) Anti-OCT4 Mouse Mouse 1:500 1:500 Santa Cruz Biotechnology Santa Cruz Biotechnology (2) Anti-OCT4(2) Anti-OCT4 Rabbit Rabbit 1:500 1:500 Stemgent Stemgent Anti-SOX2 Anti-SOX2 Rabbit Rabbit 1:1000 1:1000 Chemicon Chemicon Anti-NANOG Anti-NANOG Rabbit Rabbit 1:500 1:500 Abcam Abcam Anti-SSEA4 Anti-SSEA4 Mouse Mouse 1:500 1:500 Stemgent Stemgent Anti-TRA-1-81 Anti-TRA-1-81 Mouse Mouse 1:500 1:500 Stemgent Stemgent (Anti-p111 TUBULIN) TUJI(Anti-BIII TUJ1 TUBULIN) Mouse Mouse 1:3000 1:3000 Covance Covance Research Products ResearchProducts Anti-SMA Anti-SMA Mouse Mouse 1:500 1:500 Sigma Sigma Anti-AFP Anti-AFP Mouse Mouse 1:500 1:500 Sigma Sigma

5 5 Table 44 Summary Table of reprogramming Summary of experiments reprogrammingexperiments

TRA-1-81 TRA-1-81 Donor Cells Donor Cells Induction factors Induction factors Chemicals Chemicals Experiments Experiments positive positive colonies colonies #1 #1 17 17 DMSO #2 #2 20 20 DMSO OCT4+KLF4+SOX2+MYC OCT4+KLF4+SOX2+MYC #3 #3 23 23 #1 #1 72 72 A83+PD A83+PD #2 #2 104 104 #3 #3 91 91 #1 #1 2 2 DMSO #2 #2 33 DMSO OCT4+KLF4+SOX2 OCT4+KLF4+SOX2 #3 #3 88 #1 #1 26 26 A83+PD A83+PD #2 #2 35 35 #3 #3 44 44 NHEKs #1 #1 1 1

NHEKs (lot number: (lot number: A83+PD A83+PD #2 #2 2 2 0000087940) 0000087940) #3 #3 0 0 #1 #1 15 15 A83+PS48+PD A83+PS48+PD #2 #2 18 18 #3 #3 5 5 OCT4+KLF4 OCT4+KLF4 #1 #1 6 6 A83+VPA+PD A83+VPA+PD #2 #2 0 0 #3 #3 3 3 #1 #1 20 20 A83+NaB+PD A83+NaB+PD #2 #2 17 17 #3 #3 18 18 #1 #1 21 21 A83+PS48+NaB+PD A83+PS48+NaB+PD #2 #2 30 30

62

TRA-1-81 TRA-1-81 Cells DonorCells Donor Induction factors Induction factors Chemicals Chemicals Experiments Experiments positive positive colonies colonies #3 #3 27 27 #1 #1 4 4 OCT4 OCT4 A83+PS48+NaB+PD A83+PS48+NaB+PD #2 #2 0 0 #3 #3 33 NHEKs #1 #1 2 2 NHEKs (lot number: (lot number: OCT4 A83+PS48+NaB+PD A83+PS48+NaB+PD #2 #2 33 OCT4 2022204080 2F0661) 2F0661) #3 #3 0 0 AHEKs AHEKs OCT4 OCT4 A83+PS48+NaB+PD A83+PS48+NaB+PD #1 #1 3 3 +Par+CHIR +Par+CHIR #2 #2 2 2 #1 #1 4 4 HUVECs HUVECs OCT4 OCT4 A83+PS48+NaB+PD A83+PS48+NaB+PD #2 #2 7 7 #3 #3 4 4 HUVECs OCT4 OCT4 A83+PS48+NaB+PD A83+PS48+NaB+PD #1 #1 23 23 HUVECs +Par+CHIR +Par+CHIR #2 #2 17 17

AFDCs AFDCs OCT4 OCT4 A83+PS48+NaB+PD A83+PS48+NaB+PD #1 #1 55 +Par+CHIR +Par+CHIR #2 #2 11 11

NHEKs,

[0215] NHEKs,

[0215] Neonatal Neonatal Human Human Epidermal Epidermal Keratinocytes; Keratinocytes; HUVECs, HUVECs, Human Umbilical Human Umbilical

Cells;AHEKs, Endothelial Cells; Vein Endothelial Vein AHEKs, Adult Human Adult Human Epidermal Epidermal Keratinocytes; AFDCs, Keratinocytes;AFDCs, Amniotic Amniotic

Fluid 5 Fluid 5 Derived Derived Cells. Chemical Cells.Chemical concentration used:PD,PD, concentrationused: 0.5µM M 0.5 PD0325901; A83,A83, PD0325901; 0.5 µM pM 0.5 A- A 83-01; PS48, 83-01; PS48, 55 µM M PS48; PS48;VPA, VPA,0.5mM 0.5mM Valproic Valproic acid; acid; NaB, NaB, 0.25 0.25 mM mM Sodium Sodium butyrate; butyrate; Par,Par,

2 pM 2 Parnate; CHIR, µM Parnate; CH1R99021. CHIR,3 3µMMCHIR99021. For four-factor or or For four-factor three-factor induced three-factorinduced reprogramming, NHEKs reprogramming, NHEKs were were seeded seeded a densityofof100,000 at aat density transducedcells 100,00.0transduced per 10 cm cells per dish cm dish

and positivecolonies and positive colonieswere were counted counted four-week four-week later;later; For two-factor For two-factor inducedinduced reprogramming, reprogramming,

10 were were NHEKs 10 NHEKs seeded seeded at a density at a density of 100,000 of 100,000 transduced transduced cells cells perper1010cmcm dish andpositive dishand positive colonies counted six-week were counted colonies were Forone-factor later;For six-week later; one-factorinduced induced reprogramming,NHEKs reprogramming, and NHEKs and

AHEKs werewere AHEKs seeded seeded at a density at a density of 1,000,000 of 1,000,000 transduced cells percells transduced 10 cm 10 and perdish dish and positive cm positive colonies counted eight-week were counted colonies were later; HUVECs eight-week later; HUVECs and AFDCs andAFDCs were were seeded at at seeded a densityofof a density

200,000 transduced 200,000transduced cells perper cells 10 cm cm dish 10 dish and positive and positive colonies colonies were counted six-weeksix-week were counted later. later.

Table 15 Table 15 5 Characterization 5 Characterization of established of established human iPSC iPSC human cell lines cell lines

clone hiPSC clone hiPSC Induction Induction Cell Cell Marker Marker RT-PCR RT-PCR EB EB Teratoma Teratoma factors factors source source expression expression test test differentiation differentiation test test

hiPSC-OK#1 hiPSC-OK#1 OCT4+KLF4 OCT4+KLF4 NHEKs NHEKs '1 hiPSC-OK#3 hiPSC-OK#3 hiPSC-O#I hiPSC-O#1 'I hiPSC-O#3 hiPSC-O#3 hiPSC-O#4 hiPSC-O#4 OCT4 OCT4 NHEKs NHEKs 4 hiPSC-O#5 hiPSC-O#5 2 more 2 lines more lines

63

hiPSC-O#21 hiPSC-O#21 4 4 4 hiPSC-O#22 hiPSC-O#22 hiPSC-O#26 hiPSC-O#26 OCT4 OCT4 HUVECs HUVECs 4 4 4 hiPSC-O#31 hiPSC-O#31 4 4 4 4 7 more 7 more lines lines hiPSC-0#52 hiPSC-O#52 OCT4 OCT4 AHEKs AHEKs 4 hiPSC-O#57 hiPSC-O#57 2022204080

hiPSC-O#63 hiPSC-O#63 OCT4 OCT4 AFDCs AFDCs 4 hiPSC-O#65 hiPSC-O#65

[0216] Those

[0216] cell cell Those lines lines characterized were were characterized long-term long-term expanded for over for expanded 20 passages passages over 20 under under

hESC conventionalhESC conventional culture culture condition condition and further and further characterized characterized for marker for marker expression expression and and

whileother pluripotency;while pluripotency; linesestablished celllines othercell were establishedwere stored stored at passage 5 or56. at passage 6. Blank orBlank entries entries

indicatenot 5 indicate 5 determined. notdetermined.

DNAfingerprint Table 66 DNA Table fingerprint analysis Oct4induced analysisononOct4 induced iPSCs iPSCs andand parental cellcell parental lines lines

Genomic loci Genomic loci NHEK NHEK (pooled) (pooled) hiPSC-O#1 hiPSC-O#1 HUVEC hiPSC-O#21 hiPSC-O#21 HUVEC Amelogenin Amelogenin X, Y X,Y X, Y X,Y X X X x vWA 11, 15, 17, 18, 19 11, 15, 17, 18, 19 15,18 15, 18 15; 16 15; 16 15; 16 15; 16 vWA D8S1179 D8S1179 10,13, 16 10, 13, 16 13, 13, 10; 13 10; 13 10; 13 10; 13

TPOX 8,9,11,12 8, 9, 11, 12 8 8 8 8 8 8 TPOX FGA 19,22,23,24 19, 22, 23, 24 19,22 19, 22 24;27 24; 27 24;27 24; 27 FGA D3S1358 D3S1358 13,14,15,17 13, 14, 15, 17 17 17 14;16 14; 16 14;16 14; 16

THO1 THO1 6,7,9,9.3 6, 7, 9, 9.3 7,9 7,9 6 6 6 6

D21SI1 D21S11 24.2, 29, 30.2, 35 24.2, 29, 30.2, 35 24.2,29 24.2, 29 28; 30.2 28; 30.2 28; 30.2 28; 30.2

D18S51 D18S51 13, 14, 16, 17,18,19 13, 14, 16, 17, 18, 19 13,17 13, 17 13;18 13; 18 13;18 13; 18

Penta EE Penta 5, 8, 13, 14, 19 5, 8, 13, 14, 19 13,19 13, 19 12 12 12 12

D5S818 D5S818 8, 11, 12, 13 8, 11, 12, 13 11, 13 11, 13 12;13 12; 13 12;13 12; 13

D13S317 D13S317 8, 9, 11, 12, 13 8, 9, 11, 12, 13 9,12 9, 12 11;14 11; 14 11;14 11; 14

D7S820 D7S820 8,9,10,11 8, 9, 10, 11 9,10 9, 10 11 11 11 D16S539 D16S539 9,10,11, 12,13 9, 10, 11, 12, 13 9,13 9, 13 9; 11 9; 11 9; 11 9; 11

CSFIPO CSF1PO 10,11,12 10, 11, 12 11,12 11, 12 11;12 11; 12 11;12 11; 12

Penta DD Penta 2.2,10, 12 2.2, 10, 12 10 10 12;13 12; 13 12;13 12; 13

[0217] Fifteenpolymorphic

[02171 Fifteen shorttandem polymorphicshort repeat(STR) tandemrepeat lociloci DNA (STR)DNA thethe andand sex chromosome sexchromosome

amelogenin were marker amelogenin marker investigated. wereinvestigated.

10 10

[0218] It isunderstood

[02181 It is understood the the thatthat examples examples and embodiments and embodiments herein areherein describeddescribed for are for illustrative purposes illustrative purposes only thatvarious andthat onlyand modifications variousmodifications or changes or changes in light in light thereof will will thereof be be suggestedtotopersons suggested andarearetotobebeincluded skilledin inthetheartartand personsskilled included within the the within spirit andand spirit purview purview of of this application this scopeofofthe and scope application and appended theappended claims. claims. All publications, All publications, patents, patents, and patent and patent

5 5 cited herein applications cited applications arehereby hereinare incorporated herebyincorporated by reference by reference in their in their entirety for for entirety all all purposes. purposes. 2022204080

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SEQUENCE LISTING <110> Lin, Tongxiang Ding, Sheng The Scripps Research Institute

<120> Induction of Pluripotent Cells

<130> 014740-003810PC <140> WO PCT/US10/52896 2022204080

<141> 2010-10-15 <150> US 61/252,548 <151> 2009-10-16 <160> 47 <170> FastSEQ for Windows Version 4.0

<210> 1 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer Endo-OCT4

<400> 1 agtttgtgcc agggtttttg 20

<210> 2 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR reverse primer Endo-OCT4

<400> 2 acttcacctt ccctccaacc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer Endo-SOX2 <400> 3 caaaaatggc catgcaggtt 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer Endo-SOX2 <400> 4

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agttgggatc gaacaaaagc tatt 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer Endo-NANOG

<400> 5 tttggaagct gctggggaag 20 2022204080

<210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer Endo-NANOG <400> 6 gatgggagga ggggagagga 20 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer Endo-KLF4

<400> 7 acgatcgtgg ccccggaaaa ggacc 25

<210> 8 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR reverse primer Endo-KLF4

<400> 8 gattgtagtg ctttctggct gggctcc 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer Endo-cMYC <400> 9 gcgtcctggg aagggagatc cggagc 26 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220>

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<223> synthetic RT-PCR reverse primer Endo-cMYC <400> 10 ttgaggggca tcgtcgcggg aggctg 26 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer REX1 2022204080

<400> 11 cagatcctaa acagctcgca gaat 24

<210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer REX1 <400> 12 gcgtacgcaa attaaagtcc aga 23

<210> 13 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer UTF1

<400> 13 ccgtcgctga acaccgccct gctg 24

<210> 14 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR reverse primer UTF1 <400> 14 cgcgctgccc agaatgaagc ccac 24

<210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer TDGF2 <400> 15 ctgctgcctg aatgggggaa cctgc 25 <210> 16 <211> 27 <212> DNA

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<213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer TDGF2 <400> 16 gccacgaggt gctcatccat cacaagg 27 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence 2022204080

<220> <223> synthetic RT-PCR forward primer FGF4

<400> 17 ctacaacgcc tacgagtcct aca 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer FGF4

<400> 18 gttgcaccag aaaagtcaga gttg 24

<210> 19 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer Exo-OCT4

<400> 19 tgtctccgtc accactctgg 20

<210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer Exo-OCT4

<400> 20 atgcatgcgg atccttcg 18 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer PAX6 <400> 21 tgtccaacgg atgtgagt 18

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<210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer PAX6 <400> 22 tttcccaagc aaagatggac 20 <210> 23 2022204080

<211> 20 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer betaIII TUBULIN <400> 23 caacagcacg gccatccagg 20

<210> 24 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR reverse primer betaIII TUBULIN

<400> 24 cttggggccc tgggcctccg a 21

<210> 25 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer FOXF1

<400> 25 aaaggagcca cgaagcaagc 20

<210> 26 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR reverse primer FOXF1 <400> 26 aggctgaagc gaaggaagag g 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer HAND1

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<400> 27 tcccttttcc gcttgctctc 20

<210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer HAND1 <400> 28 2022204080

catcgcctac ctgatggacg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR forward primer AFP

<400> 29 agcagcttgg tggtggatga 20

<210> 30 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR reverse primer AFP

<400> 30 cctgagcttg gcacagatcc t 21

<210> 31 <211> 22 <212> DNA <213> Artificial Sequence

<220> <223> synthetic RT-PCR forward primer GATA6

<400> 31 tgtgcgttca tggagaagat ca 22 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic RT-PCR reverse primer GATA6 <400> 32 tttgataaga gacctcatga accgact 27 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence

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<220> <223> synthetic RT-PCR forward primer GAPDH

<400> 33 gtggacctga cctgccgtct 20

<210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> 2022204080

<223> synthetic RT-PCR reverse primer GAPDH <400> 34 ggaggagtgg gtgtcgctgt 20 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence

<220> <223> synthetic bisulfate-sequencing forward primer OCT4-1

<400> 35 ttaggaaaat gggtagtagg gattt 25

<210> 36 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> synthetic bisulfate-sequencing reverse primer OCT4-1

<400> 36 tacccaaaaa acaaataaat tataaaacct 30

<210> 37 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> synthetic bisulfate-sequencing forward primer OCT4-2 <400> 37 ggatgttatt aagatgaaga tagttgg 27

<210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> synthetic bisulfate-sequencing reverse primer OCT4-2 <400> 38 cctaaactcc ccttcaaaat ctatt 25 <210> 39 <211> 30

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<212> DNA <213> Artificial Sequence

<220> <223> synthetic bisulfate-sequencing forward primer NANOG

<400> 39 gagttaaaga gttttgtttt taaaaattat 30

<210> 40 <211> 30 <212> DNA 2022204080

<213> Artificial Sequence <220> <223> synthetic bisulfate-sequencing reverse primer NANOG <400> 40 tcccaaatct aataatttat catatctttc 30 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence

<220> <223> synthetic genotyping forward primer OCT4-Int

<400> 41 cagtgcccga aacccacac 19

<210> 42 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> synthetic genotyping forward primer OCT4-Int

<400> 42 agaggaactg cttccttcac gaca 24

<210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic genotyping forward primer SOX2-Int

<400> 43 tacctcttcc tcccactcca 20 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic genotyping reverse primer SOX2-Int <400> 44 agaggaactg cttccttcac gaca 24

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<210> 45 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> synthetic genotyping forward primer KLF4-Int

<400> 45 caccttgcct tacacatgaa gagg 24 2022204080

<210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic genotyping reverse primer KLF4-Int <400> 46 cgtagaatcg agaccgagga ga 22 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> synthetic L803 GSK3beta Inhibitor XIII

<221> PHOSPHORYLATION <222> (10)...(10) <223> phosphoserine

<221> AMIDATION <222> (11)...(11) <223> prolinamide

<400> 47 Lys Glu Ala Pro Pro Ala Pro Pro Gln Ser Pro 1 5 10

Claims (22)

Claims: 25 Jun 2025 25 Jun 2025 Claims:

1. 1. An in vitro An in vitro or or ex ex vivo vivomethod of inducing method of non-pluripotent mammalian inducing non-pluripotent mammalian cells cells intoinduced into induced pluripotent stem cells, comprising: pluripotent stem cells, comprising:

introducing intothethe introducing into non-pluripotent non-pluripotent mammalian mammalian cells at cells least at twoleast two transcription transcription factors factors selected fromthethe selected from group group consisting consisting of:Oct-3/4 of: (i) (i) Oct-3/4 andandKlf; and Klf; (ii)and (ii) Oct-3/4 Oct-3/4 and and one or oneof or more of more

Klf, Sox2, Klf, Sox2, and c-Myc;and and c-Myc; and 2022204080

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contacting contacting the the non-pluripotent non-pluripotent mammalian cellswith mammalian cells withananALK5 ALK5 inhibitor, inhibitor, a MEK a MEK

inhibitor, inhibitor,and and aaROCK inhibitor, wherein ROCK inhibitor, whereinthe theROCK ROCK inhibitor inhibitor is is presentininananamount present amount effective effective

to to increase increase efficiency efficiencyof ofreprogramming reprogramming aanon-pluripotent non-pluripotentmammalian mammaliancellcell to to a pluripotentcell, a pluripotent cell, whereinthe wherein the non-pluripotent non-pluripotentmammalian mammalian cells cells areare culturedininthe cultured theabsence absenceofoffeeder feedercells cells and underconditions and under conditions sufficient sufficient to induce to induce pluripotent pluripotent stem cells. stem cells.

2. 2. The method of claim 1, wherein introducing at least two transcription factors into the The method of claim 1, wherein introducing at least two transcription factors into the

non-pluripotent mammalian non-pluripotent mammalian cellscomprises: cells comprises: (i) (i) introducing introducing a apolynucleotide polynucleotide encoding encoding the at the at two least leasttranscription two transcription factors factors into the into the

non-pluripotent mammalian non-pluripotent mammalian cells;oror cells;

(ii) (ii)contacting contactingthe thenon-pluripotent non-pluripotentmammalian cells with mammalian cells with aa polypeptide comprisingthe polypeptide comprising the amino acid amino acid sequence sequence of at of the theleast at least two transcription two transcription factorsfactors polypeptide. polypeptide.

3. 3. The methodofofclaim The method claim1 1ororclaim claim2,2,wherein whereinthe theROCK ROCK inhibitor inhibitor hashas thethe structure: structure:

O 2 H N N (R³) N N H (R), X S R¹ (II) (II)

wherein, L2 is wherein, L² is substituted substituted or orunsubstituted unsubstitutedCC-C 1-C10 alkylene; alkylene;

y is an integer from 0 to 3; y is an integer from 0 to 3;

z is an integer from 0 to 5; Z is an integer from 0 to 5;

5 X X is is─N═, -N=,─CH═ or ─CR -CH= or -CR=; ═; R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted

heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted

heterocycloalkyl, substituted heterocycloalkyl, substituted or unsubstituted or unsubstituted aryl, aryl, or substituted or substituted or unsubstituted or unsubstituted heteroaryl; heteroaryl;

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R , RR4 and andRR5 are areindependently ─CN,-CN, ─S(O)nR 6 7 8 , ─NR-NRR, R , ─C(O)R 9 , ─NR 10 ─C(O)R11, 25 Jun 2025 Jun 2025 R³, independently -S(O)nR, -C(O)R, -NR¹-C(O)R¹,

─NR12─C(O)-OR¹³, -NR¹²-C(O) ─OR13, -C(O)NR¹R¹, ─C(O)NR14R15-NR¹S(O)R¹, , ─NR16S(O)2R-OR¹, 17 , ─OR18 , ─S(O)2NR -S(O)NR¹, 19 , substituted substituted or or

unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted

cycloalkyl, substituted cycloalkyl, substituted or or unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl, or aryl, or 2022204080 25

substituted orunsubstituted substituted or unsubstituted heteroaryl, heteroaryl, wherein wherein n integer n is an is an integer from 0 from 0 to 2, if to 2, wherein wherein if z is greater Z is greater

3 than 1, two R moieties are optionally joined together to form a substituted or unsubstituted than 1, two R³ moieties are optionally joined together to form a substituted or unsubstituted

cycloalkyl, substitutedor or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or aryl, or 2022204080

cycloalkyl, substituted unsubstituted heterocycloalkyl, substituted or unsubstituted

substituted orunsubstituted substituted or unsubstituted heteroaryl; heteroaryl; and and

R,6,R, R R7R, 8 R¹, , R9, RR¹¹, , RR, 10 11 , R12 , RR¹², , R13R¹, R¹³, 14 R¹, , R15, RR¹, , RR¹, 16 R¹17and 18 , R , R R¹and 19 areRindependently are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,

unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or

unsubstituted aryl, or substituted or unsubstituted heteroaryl; unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or a racemate, or a racemate,diastereomer, diastereomer, tautomer, tautomer, or a geometric or a geometric isomer or isomer thereof, thereof, a or a pharmaceutically acceptable salt thereof. pharmaceutically acceptable salt thereof.

4. 4. The method The methodofofclaim claim3,3,wherein: wherein: L²2 is L is methylene; methylene;

X is N═ X is or CH═; N= or CH=; R¹1 is R is hydrogen; or hydrogen; or

y and z are 0. y and Z are 0.

5. 5. The method The methodofofclaim claim3,3,wherein whereinthe theROCK ROCK inhibitor inhibitor has has the the structure: structure:

O ZI H N IZ N N N H N S , ,

O H N IZ N N N H S ,, or or

O ZI H N IZ N N N H S N OCH3 .

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6. The method methodofofany anyone oneofofclaims claims1 1toto5,5,wherein whereinthe thenon-pluripotent non-pluripotentmammalian mammalian cells areare 25 Jun 2025 2022204080 25 Jun 2025

6. The cells

humancells. human cells.

7. 7. The method The methodofofany anyone oneofofclaims claims1 1toto6,6,further further comprising comprisingaaglycogen glycogensynthase synthasekinase kinase3 3 (GSK3) inhibitor, aa histone (GSK3) inhibitor, histone deacetylase (HDAC) deacetylase (HDAC) inhibitor,ororboth. inhibitor, both.

8. 8. The method The methodofofany anyone oneofofclaims claims1 1toto7,7,wherein wherein 2022204080

(i) (i)the theALK5 inhibitor is ALK5 inhibitor is A-83-01 or SB431542, A-83-01 or SB431542, oror

(ii) (ii)the theMEK inhibitor is MEK inhibitor isPD0325901. PD0325901.

9. 9. The method The methodofofany anyone oneofofclaims claims1 1toto8,8,further further comprising comprisingaamolecular moleculartether, tether, wherein wherein the molecular tether is capable of attaching the non-pluripotent mammalian cells directly to a the molecular tether is capable of attaching the non-pluripotent mammalian cells directly to a

solid culture surface, solid culture surface,

whereinthe wherein the molecular moleculartether tether increases increases efficiency efficiency of of reprogramming thenon-pluripotent reprogramming the non-pluripotent mammalian mammalian cellstotoinduced cells inducedpluripotent pluripotentstem stemcells. cells.

10. 10. A mixture A mixture comprising: comprising:

(a) (a) one or more one or mammalian more mammalian cells; cells;

(b) (b) a Rho a Rho kinase(ROCK) kinase (ROCK) inhibitor inhibitor

(c) (c) an an ALK5 ALK5 inhibitor;and inhibitor; and (d) (d) a MAP/ERK a MAP/ERK kinase kinase (MEK) (MEK) inhibitor; inhibitor;

whereinthe wherein the ROCK ROCK inhibitor inhibitor is is presentininananamount present amount effectivetotoincrease effective increaseefficiency efficiency of of reprogramming reprogramming a non-pluripotent a non-pluripotent mammalian mammalian cell cell to atopluripotent a pluripotent mammalian mammalian cell, cell, and and whereinthe wherein the mixture mixturedoes doesnot notcomprise comprisefeeder feedercells. cells.

11. 11. The The mixture mixture of claim of claim 10, wherein 10, wherein the ROCK the ROCK inhibitor inhibitor has thehas the structure: structure:

O H N N N N H N S , O ZI H N IZ N N N H S ,, or or

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O ZI H N IZ N N N H OCH . N S

12. 12. A composition A composition comprising: comprising:

(a) (a) a Rho a Rho kinase(ROCK) kinase (ROCK) inhibitor; inhibitor;

(b) (b) an an ALK5 ALK5 inhibitor; inhibitor; 2022204080

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(c) (c) a MAP/ERK a MAP/ERK kinase kinase (MEK) (MEK) inhibitor; inhibitor; andand

(d) (d) a moleculartether; a molecular tether; whereinthe wherein the ROCK ROCK inhibitor inhibitor is is presentininananamount present amount effectivetotoincrease effective increaseefficiency efficiency of of reprogramming reprogramming a non-pluripotent a non-pluripotent mammalian mammalian cell cell to atopluripotent a pluripotent mammalian mammalian cell, cell, and and wherein the molecular tether is capable of attaching a mammalian cell directly to a solid wherein the molecular tether is capable of attaching a mammalian cell directly to a solid

culture surface. culture surface.

13. 13. The The composition composition of claim of claim 12, wherein 12, wherein the ROCK the ROCK inhibitor inhibitor has thehas the structure: structure:

O 2 ZI H N IZ N N N H (R³) (R)y X S R¹ (II) (II)

wherein, L2 is wherein, L² is substituted substituted or orunsubstituted unsubstitutedCC-C 1-C10 alkylene; alkylene;

y is an integer from 0 to 3; y is an integer from 0 to 3;

z is an integer from 0 to 5; Z is an integer from 0 to 5;

5 X is -N=,-CH=or X is ─N═, ─CH═ or ─CR -CR= -N=, ═; or -CR=;

R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted

heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted

heterocycloalkyl, substituted heterocycloalkyl, substituted or unsubstituted or unsubstituted aryl, aryl, or substituted or substituted or unsubstituted or unsubstituted heteroaryl; heteroaryl;

3 R R³, , RR4 and andRR5 are areindependently ─CN,-CN, independently ─S(O)nR 6 7 8 , ─NR-NRR, -S(O)nR, R , ─C(O)R 9 -C(O)R, , ─NR 10 ─C(O)R11, -NR¹-C(O)R¹,

─NR12─C(O)-OR¹³, -NR¹²-C(O) ─OR13, -C(O)NR¹R¹, ─C(O)NR14R15-NR¹S(O)R¹, , ─NR16S(O)2R-OR¹, 17 , ─OR18 , ─S(O)2NR -S(O)NR¹, 19 , substituted substituted oror

unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted

cycloalkyl, substituted cycloalkyl, substituted or or unsubstituted unsubstituted heterocycloalkyl, heterocycloalkyl, substituted substituted or unsubstituted or unsubstituted aryl, or aryl, or

substituted orunsubstituted substituted or unsubstituted heteroaryl, heteroaryl, wherein wherein n integer n is an is an integer from 0 from 0 to 2, if to 2, wherein wherein if z is greater Z is greater

3 than 1, two R moieties are optionally joined together to form a substituted or unsubstituted than 1, two R³ moieties are optionally joined together to form a substituted or unsubstituted

69 cycloalkyl, substitutedor or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or aryl, or 25 Jun 2025 2022204080 25 Jun 2025 cycloalkyl, substituted unsubstituted heterocycloalkyl, substituted or unsubstituted substituted orunsubstituted substituted or unsubstituted heteroaryl; heteroaryl; and and

R,6,R, R R7R, 8 R¹, , R9, RR¹¹, , RR, 10 11 , R12 , RR¹², , R13R¹, R¹³, 14 R¹, , R15, RR¹, , RR¹, 16 R¹17and 18 , R , R R¹and 19 areRindependently are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,

unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or

unsubstituted aryl, or substituted or unsubstituted heteroaryl; unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or a racemate, racemate,diastereomer, diastereomer, tautomer, or a geometric isomer or thereof, or a 2022204080

or a tautomer, or a geometric isomer thereof, a

pharmaceutically acceptable salt thereof. pharmaceutically acceptable salt thereof.

14. 14. The The composition composition of claim of claim 13, wherein: 13, wherein:

L²2 is L is methylene; methylene;

X is N═ X is N= or or CH═; CH=; R¹1 is R is hydrogen; or hydrogen; or

y and z are 0. y and Z are 0.

15. 15. The The composition composition of claim of claim 13, wherein 13, wherein the ROCK the ROCK inhibitor inhibitor has thehas the structure: structure:

O ZI H N IZ N N N H N S , ,

O ZI H N IZ N N N H S ,, or or

O H N IZ N N N H S N OCH .

16. 16. The The composition composition of one of any anyof one of claims claims 12 to12 to further 15, 15, further comprising comprising a glycogen a glycogen synthase synthase

kinase 33 (GSK3) kinase inhibitor, aa histone (GSK3) inhibitor, histone deacetylase (HDAC) deacetylase (HDAC) inhibitor,ororboth. inhibitor, both.

17. 17. The The composition composition of one of any anyof one of claims claims 12 to12 to wherein: 16, 16, wherein: (i) (i)the theALK5 inhibitor is ALK5 inhibitor is A-83-01 or SB431542; A-83-01 or SB431542; oror

(ii) (ii)the theMEK inhibitor is MEK inhibitor isPD0325901. PD0325901.

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18. The The composition of one anyof one of claims 12 to12 to wherein 17, wherein the molecular tethertether is selected 25 Jun 2025 Jun 2025 18. composition of any claims 17, the molecular is selected

from the group from the groupconsisting consisting of of matrigel, matrigel, an an extracellular extracellularmatrix matrix(ECM) or ECM (ECM) or ECM analog, analog, laminin, laminin,

fibronectin, andcollagen. fibronectin, and collagen.

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19. 19. The The composition composition of one of any anyof one of claims claims 14-18, 14-18, further further comprising comprising one orone or mammalian more more mammalian cells. cells. 2022204080

20. The The 20. composition composition of claim of claim 19, wherein 19, wherein theorone the one or more more mammalian mammalian cells comprise cells comprise non- non- pluripotent cells or pluripotent cells. pluripotent cells or pluripotent cells.

21. Use Use 21. of aof a composition composition in induction in induction of non-pluripotent of non-pluripotent mammalian mammalian cellsainto cells into a pluripotent pluripotent

mammalian mammalian cell,wherein cell, wherein thecomposition the composition comprises: comprises:

(a) (a) a Rho a Rho kinase(ROCK) kinase (ROCK) inhibitor inhibitor

(b) (b) an an ALK5 ALK5 inhibitor;and inhibitor; and (c) (c) a MAP/ERK a MAP/ERK kinase kinase (MEK) (MEK) inhibitor; inhibitor;

whereinthe wherein the ROCK ROCK inhibitor inhibitor is is presentininananamount present amount effectivetotoincrease effective increaseefficiency efficiency of of reprogramming a non-pluripotent reprogramming a non-pluripotent mammalian mammalian cell cell to atopluripotent a pluripotent mammalian mammalian cell. cell.

22. The The 22. useclaim use of of claim 21, 21, wherein wherein the ROCK the ROCK inhibitor inhibitor has thehasstructure: the structure: O ZI H N IZ N N N H N S ,,

O H N N N N H S ,, or or

O ZI H N IZ N N N H S N OCH .

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